Antibacterial phage, phage peptides and methods of use thereof

ABSTRACT

The present invention is directed to the field of phage therapy for the treatment and control of bacterial infections. In particular, the present invention is directed to the novel bacteriophages F1245/05, F168/08, F170/08, F770/05, F197/08, F86/06, F87s/06 and F91a/06, isolated polypeptides thereof, compositions comprising one or more of the novel bacteriophages and/or isolated polypeptides and methods for the treatment and prevention of bacterial infection, either alone or in combination with other antibacterial therapies, e.g., antibiotics or other phage therapies.

1. RELATED APPLICATIONS

This International Application claims priority to U.S. ProvisionalApplication Ser. No. 61/150,585, filed Feb. 6, 2009, entitled“Antibacterial Phage and Uses Thereof” and to U.S. ProvisionalApplication Ser. No. 61/218,345, filed Jun. 18, 2009, entitled“Antibacterial Phage, Phage Peptides and Methods of Use Thereof”. Wherepermitted, the subject matter and contents, including sequence listing,of these provisional applications are incorporated by reference hereinin their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 19, 2014, isnamed 16395-105001_SL.txt and is 1,767,167 bytes in size.

2. FIELD OF THE INVENTION

The present invention is directed to the field of phage therapy for thetreatment and control of bacterial infections. In particular, thepresent invention is directed to the novel bacteriophages F1245/05,F168/08, F170/08, F770/05, F197/08, F86/06, F87s/06 and F91a/06,isolated polypeptides thereof, compositions comprising one or more ofthe novel bacteriophages and/or isolated polypeptides and methods forthe treatment and prevention of bacterial infections caused byAcinetobacter baumannii, Enterococcus faecalis, E. faecium, Pseudomonasaeruginosa, and/or Staphylococcus aureus, either alone or in combinationwith other antibacterial therapies, e.g., antibiotics or other phagetherapies.

3. BACKGROUND

Bacteriophages (phage) are viruses that specifically infect and lysebacteria. Phage therapy, a method of using whole phage viruses for thetreatment of bacterial infectious diseases, was introduced in the 1920sby Felix d'Herelle. Initially, phage therapy was vigorously investigatedand numerous studies were undertaken to assess the potential of phagetherapy for the treatment of bacterial infection in humans and animals.Early success prompted the development of multiple commercial phagepreparations. For example, in 1940 Eli Lilly Company produced 7 phageproducts for human use, including phage preparations for treatingdifferent sicknesses caused by Staphylococcus sp., E. coli and otherpathogenic bacteria. These preparations were used to treat infectionsthat cause abscesses, purulent wounds, vaginitis, acute chronicupper-respiratory tract infections and mastoid infections.

However, with the development of antibiotics in the 1940s, interest inphage-based therapeutics declined in the Western world. One of the mostimportant factors that contributed to this decline was the lack ofstandardized testing protocols and methods of production. The failure todevelop industry wide standards for the testing of phage therapiesinterfered with the documentation of study results, leading to aperceived lack of efficacy as well as problems of credibility regardingthe value of phage therapy. Further, problems related to the productionof phage samples/specimens complicated initial study and research.Diverse stabilizers and preservatives were initially used in attempts toincrease the viability of the phage therapeutics. However, because thebiology of both the phage and the various stabilizers were poorlyunderstood, many of the ingredients added in an attempt to prolong theviability of phage preparations proved to be either toxic to humans orto negatively impact long term storage. Another problem related to phageproduction was the purity grade of the commercial preparations of theseviruses. At the time, phage therapy preparations generally consisted ofraw lysates of host bacteria that had been treated with the phage ofinterest. Thus, many preparations contained what are now recognized asundesired bacterial components, e.g., endotoxins. Accordingly, adverseevents were often associated with the preparations, particularly inpatients receiving them intravenously. Nevertheless, in Eastern Europeand the former Soviet Union, where access to antibiotics was limited,the development and use of phage therapy continued jointly with, or inplace of, antibiotics.

With the rise of antibiotic resistant strains of bacteria, however,interest in phage-based therapeutics has returned in the Western world.Even though novel classes of antibiotics may be developed, the prospectthat bacteria will eventually develop resistance to the new drugs hasintensified the search for non-chemotherapeutic means for controlling,preventing, and treating bacterial infections. There are three mainphage-based strategies for using phage therapy in a clinicalenvironment: 1) the administration of virulent phages; 2) the use ofendolysins or purified lysins encoded by bacteriophages 3) the use ofstructural proteins of the identified phages as metabolic inhibitors ofkey enzymes for the synthesis of bacterial peptidoglycan.

There is therefore a need to develop novel bacteriophages and phageproducts as potential therapeutic and prophylactic agents for use invivo to eliminate pathogenic bacteria. In particular, there is a needfor bacteriophages capable of lysing nosocomial bacteria, includingAcinetobacter baumannii, Enterococcus faecalis, E. faecium, Pseudomonasaeruginosa, and/or Staphylococcus aureus. Because most phage and phagepeptides studied to date exhibit activity often restricted to therelated species, or subspecies, of bacteria from which they areisolated, the novel phage-based therapies may find particular use in thehospital setting, selectively targeting nosocomial pathogens withoutaffecting the normal surrounding flora.

4. SUMMARY OF THE INVENTION

The present invention is directed to isolated bacteriophages and toisolated antibacterial polypeptides of bacteriophage origin for thetreatment, prevention, or management of conditions associated withinfection by Gram-positive or Gram-negative bacteria. In particular, theisolated bacteriophage or polypeptides of the invention may be used inpharmaceutical compositions for the treatment, prophylaxis, ormanagement of infection by nosocomial pathogens, e.g., Gram-positivebacteria including but not limited to Enterococcus faecalis, E. faecium,E. hirae, E. avium, Staphylococcus aureus, S. epidermidis, S.auricularis, S. capitis, S. haemolyticus, S. hominis, S. saprophyticus,S. simulans, and S. xylosis; and/or Gram-negative bacteria including butnot limited to Acinetobacter baumannii, and Pseudomonas aeruginosa. Incertain embodiments, the pharmaceutical compositions of the inventionare of use in the treatment of conditions associated with infection byantibiotic resistant strains of bacteria, e.g., methicillin resistantstrains of Staphylococcus aureus (MRSA). In particular embodiments, theisolated bacteriophages or polypeptides of the invention are used forthe topical treatment of infection by nosocomial pathogens in a subjectin need thereof. In other embodiments, the isolated bacteriophages orpolypeptides of the invention are used for the diagnosis of theinfective agent in a sample (e.g., tissue, blood, urine, sputum sample)derived from a patient. In other embodiments, the isolatedbacteriophages or polypeptides of the invention are used as aprophylactic disinfectant or anti-infective for the preparation of solidsurfaces, including skin or other epidermal surfaces.

In certain embodiments, the invention provides an isolatedbacteriophage, F168/08 or F170/08, having a genome comprising thenucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2, respectively, andexhibiting antibacterial activity against one or more strains ofEnterococcus faecalis and/or E. faecium. In other embodiments, theinvention provides an isolated bacteriophage, F770/05, having a genomecomprising the nucleic acid sequence of SEQ ID NO:3 and exhibitingantibacterial activity against one or more strains of Pseudomonasaeruginosa. In yet other embodiments, the invention provides theisolated bacteriophage F197/08, F86/06, F87s/06 or F91a/06 having agenome comprising the nucleic acid sequence of SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6 or SEQ ID NO:7, respectively, and exhibiting antibacterialactivity against one or more strains of Staphylococcus aureus. In stillyet other embodiments, the invention provides an isolated bacteriophage,F1245/05, having a genome comprising the nucleic acid sequence of SEQ IDNO:760 and exhibiting antibacterial activity against one or more strainsof Acinetobacter baumannii.

The invention also encompasses isolated bacteria infected with one ormore bacteriophage of the invention. In specific embodiments, theinvention provides an isolated E. faecalis infected with a bacteriophagehaving a genome comprising or consisting of the nucleic acid sequence ofSEQ ID NO:1 and/or SEQ ID NO:2. In other embodiments, the inventionprovides an isolated E. faecium infected with a bacteriophage having agenome comprising or consisting of the nucleic acid sequence of SEQ IDNO:1 and/or SEQ ID NO:2. In still other embodiments, the inventionprovides an isolated P. aeruginosa infected with a bacteriophage havinga genome comprising or consisting of the nucleic acid sequence of SEQ IDNO:3. In yet other embodiments, the invention provides an isolated S.aureus infected with one or more bacteriophages having a genomecomprising or consisting of the nucleic acid sequence of SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6 and/or SEQ ID NO:7. In still yet otherembodiments, the invention provides an isolated A. baumannii infectedwith a bacteriophage having a genome comprising or consisting of thenucleic acid sequence of SEQ ID NO:760.

The present invention encompasses polypeptides isolated frombacteriophage F1245/05, F168/08, F170/08, F770/05, F197/08, F86/06,F87s/06 and/or F91a/06, which polypeptides exhibit antibacterialactivity against one or more species or strains of Gram-positive orGram-negative bacterium, e.g., A. baumannii, E. faecalis, E. faecium, P.aeruginosa and/or S. aureus. In specific embodiments, the polypeptidesof the invention isolated or derived from F168/08 or F/170/08 exhibitantibacterial or antimicrobial activity, e.g., lytic killing activity,against at least E. faecalis and/or E. faecium; those isolated orderived from F770/05 against at least P. aeruginosa; those isolated orderived from F197/08, F86/06, F87s/06 or F91a/06 against at least S.aureus; and those isolated or derived from F1245/05 against at least A.baumannii.

In certain embodiments, a polypeptide of the invention comprises orconsists of an isolated endolysin or fragment thereof (e.g., a CHAPdomain) that exhibits antibacterial activity against one or more speciesor strains of bacteria, e.g., Gram-positive bacteria and/orGram-negative bacteria such as A. baumannii, E. faecalis, E. faecium, P.aeruginosa and/or S. aureus. In specific embodiments, the polypeptide ofthe invention is an isolated lysin protein, e.g., an endolysin or taillysin, comprising or consisting of the amino acid sequence SEQ ID NO:68,SEQ ID NO:184, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:446, SEQ IDNO:447, SEQ ID NO:448, SEQ ID NO:575, SEQ ID NO:641 or SEQ ID NO:712. Inyet still other embodiments, the invention provides a polypeptidecomprising or consisting of an amino acid sequence selected from thegroup consisting of SEQ ID NO: 761 to SEQ ID NO: 816.

In other embodiments, a polypeptide of the invention comprises afragment, variant or derivative of SEQ ID NO:68, SEQ ID NO:184, SEQ IDNO:202, SEQ ID NO:203, SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQID NO:575, SEQ ID NO:641 or SEQ ID NO:712, wherein said fragment,variant or derivative has antibacterial activity or antimicrobialactivity, e.g., lytic killing activity, against one or more strains ofE. faecalis, E. faecium, P. aeruginosa and/or S. aureus. In specificexamples in accordance with this embodiment, the variant, fragment orderivative of the amino acid sequence of SEQ ID NO:68, SEQ ID NO:184,SEQ ID NO:202 and/or SEQ ID NO:203 exhibits antibacterial orantimicrobial activity (e.g., lytic killing activity) against one ormore strains of E. faecalis and/or E. faecium. In other examples inaccordance with this embodiment, the variant, fragment or derivative ofthe amino acid sequence of SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448,SEQ ID NO:575, SEQ ID NO:641 and/or SEQ ID NO:712 exhibits antibacterialor antimicrobial activity (e.g., lytic killing activity) against one ormore strains of S. aureus.

In specific embodiments, the isolated polypeptide of the inventioncomprises or consists of the CHAP domain of SEQ ID NO:68, SEQ ID NO:446,SEQ ID NO:575, SEQ ID NO:641 or SEQ ID NO:712. In certain embodiments,the isolated polypeptide comprises or consists of the CHAP domain of SEQID NO:68, SEQ ID NO:446, SEQ ID NO:575, SEQ ID NO:641 or SEQ ID NO:712,e.g., having the amino acid sequence of SEQ ID NO:755, SEQ ID NO:756,SEQ ID NO:757, SEQ ID NO:758 or SEQ ID NO:759, respectively. In otherembodiments, a polypeptide of the invention comprises a fragment,variant or derivative of SEQ ID NO:755, SEQ ID NO:756, SEQ ID NO:757,SEQ ID NO:758 or SEQ ID NO:759, wherein said fragment, variant orderivative has antibacterial activity or antimicrobial activity, e.g.,lytic killing activity, against at least one or more strains of E.faecalis, E. faecium, P. aeruginosa and/or S. aureus. In yet still otherembodiments, a polypeptide of the invention comprises a fragment,variant or derivative of SEQ ID NO: 761 to SEQ ID NO: 816, wherein saidfragment, variant or derivative has antibacterial activity orantimicrobial activity, e.g., lytic killing activity, against at leastone or more strains of A. baumannii.

In other embodiments, a polypeptide of the invention comprises orconsists of an isolated tail length tape measure protein or tail protein(e.g., tail component, tail fiber protein, adsorption associated tailprotein), or fragment thereof, having a biologic function associatedwith the bacteriophage from which it is derived, e.g., antimicrobial orantibacterial activity (e.g., lytic killing activity), which function isdirected against at least one or more species or strains of E. faecalis,E. faecium, P. aeruginosa, S. aureus, and/or A. baumannii. In specificembodiments, the polypeptide of the invention is an isolated tail lengthtape measure protein or tail proteins comprising or consisting of theamino acid sequence SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:204, SEQ IDNO:214, SEQ ID NO:435, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:525, SEQID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530,SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ IDNO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQID NO:567, SEQ ID NO:568, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:700,SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704 or SEQ IDNO:795. In other embodiments, a polypeptide of the invention comprises afragment, variant or derivative of SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:204, SEQ ID NO:214, SEQ ID NO:435, SEQ ID NO:438, SEQ ID NO:440, SEQID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529,SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ IDNO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQID NO:539, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:632, SEQ ID NO:633,SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ IDNO:704 or SEQ ID NO:795, wherein said fragment, variant or derivativeexhibits a biologic function associated with the bacteriophage fromwhich it is derived, e.g., antimicrobial or antibacterial activity(e.g., lytic killing activity), which function is directed against oneor more strains of E. faecalis, E. faecium, P. aeruginosa, S. aureus,and/or A. baumannii.

In certain embodiments, the invention encompasses a variant, fragment orderivative of the amino acid sequence of SEQ ID NO:61 or SEQ ID NO:63that exhibits a biologic function associated with the bacteriophagehaving a genome comprising or consisting of the nucleic acid sequenceSEQ ID N0:1, e.g., antimicrobial or antibacterial activity (e.g., lytickilling activity), which function is directed against one or morestrains of E. faecalis and/or E. faecium. In other embodiments, theinvention encompasses a variant, fragment or derivative of the aminoacid sequence of SEQ ID NO:204 or SEQ ID NO:214 that exhibits a biologicfunction associated with the bacteriophage having a genome comprising orconsisting of the nucleic acid sequence SEQ ID NO:2, e.g., antimicrobialor antibacterial activity (e.g., lytic killing activity), which functionis directed against one or more strains of E. faecalis and/or E.faecium.

In certain embodiments, the invention encompasses a variant, fragment orderivative of the amino acid sequence of SEQ ID NO:435 or SEQ ID NO:438that exhibits a biologic function associated with the bacteriophagehaving a genome comprising or consisting of the nucleic acid sequenceSEQ ID NO:3, e.g., antimicrobial or antibacterial activity (e.g., lytickilling activity), which function is directed against one or morestrains of P. aeruginosa.

In certain embodiments, the invention encompasses a variant, fragment orderivative of the amino acid sequence of SEQ ID NO:440, SEQ ID NO:525,SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ IDNO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538 or SEQ ID NO:539that exhibits a biologic function associated with the bacteriophagehaving a genome comprising or consisting of the nucleic acid sequenceSEQ ID NO:4, e.g., antimicrobial or antibacterial activity (e.g., lytickilling activity), which function is directed against one or morestrains of S. aureus. In other embodiments, the invention encompasses avariant, fragment or derivative of the amino acid sequence of SEQ IDNO:567 or SEQ ID NO:568 that exhibits a biologic function associatedwith the bacteriophage having a genome comprising or consisting of thenucleic acid sequence SEQ ID NO:5, e.g., antimicrobial or antibacterialactivity (e.g., lytic killing activity), which function is directedagainst one or more strains of S. aureus. In yet other embodiments, theinvention encompasses a variant, fragment or derivative of the aminoacid sequence of SEQ ID NO:632 or SEQ ID NO:633 that exhibits a biologicfunction associated with the bacteriophage having a genome comprising orconsisting of the nucleic acid sequence SEQ ID NO:6, e.g., antimicrobialor antibacterial activity (e.g., lytic killing activity), which functionis directed against one or more strains of S. aureus. In yet otherembodiments, the invention encompasses a variant, fragment or derivativeof the amino acid sequence of SEQ ID NO:700, SEQ ID NO:701, SEQ IDNO:702, SEQ ID NO:703 or SEQ ID NO:704 that exhibits a biologic functionassociated with the bacteriophage having a genome comprising orconsisting of the nucleic acid sequence SEQ ID NO:7, e.g., antimicrobialor antibacterial activity (e.g., lytic killing activity), which functionis directed against one or more strains of S. aureus.

In certain embodiments, the invention encompasses a variant, fragment orderivative of the amino acid sequence of SEQ ID NOS: 761-816 thatexhibits a biologic function associated with the bacteriophage having agenome comprising or consisting of the nucleic acid sequence SEQ IDNO:760, e.g., antimicrobial or antibacterial activity (e.g., lytickilling activity), which function is directed against one or morestrains of A. baumannii.

In certain embodiments, the invention provides for isolated polypeptidesthat exhibit antimicrobial or antibacterial activity (e.g., lytickilling activity) against one or more strains of bacteria (e.g.,Gram-positive bacteria (e.g., E. faecalis, E. faecium, S. aureus),Gram-negative bacteria (e.g., of A. baumannii, P. aeruginosa) orbacteria not classified as either Gram-positive or Gram-negative),wherein the isolated polypeptides have an amino acid sequence with atleast 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity to asecond amino acid sequence of the same length (i.e., consisting of thesame number of residues), which second amino acid sequence is SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:68, SEQ ID NO:184, SEQ ID NO:202, SEQ IDNO:203, SEQ ID NO:204, SEQ ID NO:214, SEQ ID NO:435, SEQ ID NO:438, SEQID NO:440, SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:525,SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ IDNO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539,SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:575, SEQ ID NO:632, SEQ IDNO:633, SEQ ID NO:641, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQID NO:703, SEQ ID NO:704, SEQ ID NO:712, SEQ ID NO:755, SEQ ID NO:756,SEQ ID NO:757, SEQ ID NO:758, SEQ ID NO:759, SEQ ID NOS:761-816, and/ora fragment thereof.

The invention further provides isolated polypeptides comprising orconsisting of the amino acid sequence of any of SEQ ID NOS:8-130, SEQ IDNOS:131-343, SEQ ID NOS:344-438, SEQ ID NOS:439-553, SEQ ID NOS:554-616,SEQ ID NOS:617-681, SEQ ID NOS:682-759, and SEQ ID NOS:761-816. In otherembodiments, isolated polypeptides of the invention recombinantly fusedor chemically conjugated (e.g., covalent or non-covalent conjugation) totherapeutic agents (e.g., heterologous polypeptides or small molecules)are provided.

The invention also encompasses polynucleotides that encode thepolypeptides of the invention. In a specific embodiment, the inventionprovides an isolated nucleic acid comprising a nucleic acid sequenceencoding the polypeptide of any of SEQ ID NOS:8-130, SEQ ID NOS:131-343,SEQ ID NOS:344-438, SEQ ID NOS:439-553, SEQ ID NOS:554-616, SEQ IDNOS:617-681, SEQ ID NOS:682-759, and SEQ ID NOS 761-816. In otherembodiments, the invention provides an isolated nucleic acid comprisinga nucleic acid sequence encoding the polypeptide of any of SEQ ID NO:61,SEQ ID NO:63, SEQ ID NO:68, SEQ ID NO:184, SEQ ID NO:202, SEQ ID NO:203,SEQ ID NO:204, SEQ ID NO:214, SEQ ID NO:435, SEQ ID NO:438, SEQ IDNO:440, SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:525, SEQID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530,SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ IDNO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQID NO:567, SEQ ID NO:568, SEQ ID NO:575, SEQ ID NO:632, SEQ ID NO:633,SEQ ID NO:641, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ IDNO:703, SEQ ID NO:704, SEQ ID NO:712, SEQ ID NO:755, SEQ ID NO:756, SEQID NO:757, SEQ ID NO:758, SEQ ID NO:759, SEQ ID NOS 761-816, or activefragment, variant or derivative thereof, which polypeptide or activefragment, variant or derivative exhibits a biologic function associatedwith the bacteriophage from which it is isolated and/or derived, e.g.,antimicrobial or antibacterial activity (e.g., lytic killing activity).The invention also relates to a vector comprising said nucleic acid. Inone specific embodiment, said vector is an expression vector. Theinvention further provides host cells containing a vector comprisingpolynucleotides encoding the polypeptides of the invention.

The present invention encompasses methods for the production ofpolypeptides of the invention or active fragments thereof, in particularfor use in pharmaceutical compositions, i.e., antimicrobialcompositions. For example, the polypeptides of the invention may beisolated directly from cell cultures (e.g., bacterial cell cultures)infected with bacteriophage F1245/05, F168/08, F170/08, F770/05,F197/08, F86/06, F87s/06 or F91a/06. Alternatively, the polypeptides ofthe present invention may be derived by recombinant means usingexpression vectors comprising nucleic acid sequence encodingpolypeptides of the invention, e.g., SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:68, SEQ ID NO:184, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQID NO:214, SEQ ID NO:435, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:446,SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:525, SEQ ID NO:526, SEQ IDNO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536,SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:567, SEQ IDNO:568, SEQ ID NO:575, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:641, SEQID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704,SEQ ID NO:712, SEQ ID NO:755, SEQ ID NO:756, SEQ ID NO:757, SEQ IDNO:758, SEQ ID NO:759, SEQ ID NOS:761-816, or active fragments,derivatives or variants thereof. The polypeptides of the invention orfragments thereof can be produced by any method known in the art for theproduction of a polypeptide, in particular, by chemical synthesis or byrecombinant expression techniques. In specific embodiments, theinvention relates to a method for recombinantly producing a phageprotein, e.g., a lysin protein, tail protein or active fragment, variantor derivative thereof, said method comprising: (i) culturing underconditions suitable for the expression of said protein in a medium, ahost cell containing a vector comprising a nucleic acid sequenceencoding the amino acid sequence SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:68, SEQ ID NO:184, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQID NO:214, SEQ ID NO:435, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:446,SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:525, SEQ ID NO:526, SEQ IDNO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536,SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:567, SEQ IDNO:568, SEQ ID NO:575, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:641, SEQID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704,SEQ ID NO:712, SEQ ID NO:755, SEQ ID NO:756, SEQ ID NO:757, SEQ IDNO:758, SEQ ID NO:759, SEQ ID NOS:761-816, or fragment thereof; and (ii)recovery of said protein from said medium. In certain embodiments, thenucleic acid sequence encoding the polypeptide of the invention isoperably linked to a heterologous promoter.

The invention also encompasses methods for the diagnosis of thecausative agent in a clinical presentation of bacterial infection. Theisolated bacteriophages or polypeptides of the invention may be used toaid in the determination of species of bacteria in a patient sample byestablishing susceptibility of the bacteria in the sample to thebacteriophages and/or polypeptides of the invention. Such methodsfurther encompass methods of evaluation of antibacterial activity of theisolated bacteriophages and/or polypeptides of the invention.Antibacterial activity of the bacteriophages or the polypeptides of theinvention, or susceptibility of an unknown sample to such activity, maybe assessed by any method known in the art and/or described herein. Incertain embodiments, antibacterial activity and/or susceptibility isassessed by culturing known bacteria and/or patient tissue, blood, fluidor swab samples according to standard techniques (e.g., in liquidculture or on agar plates), contacting the culture with bacteriophagesand/or polypeptides of the invention and monitoring cell growth aftersaid contacting. For example, in a liquid culture, the bacteria (e.g.,A. baumannii, E. faecalis, E. faecium, P. aeruginosa, S. aureus) may begrown to a optical density (“OD”) representative of a mid-point inexponential growth of the culture; the culture is exposed to one or moreconcentrations of one or more bacteriophages and/or polypeptides of theinvention and the OD is monitored relative to a control culture.Decreased OD relative to a control culture is representative of abacteriophage and/or polypeptide exhibiting antibacterial activity(e.g., exhibits lytic killing activity) against the tested sample orbacterial species and/or strain in the culture. Similarly, bacterialcolonies can be allowed to form on an agar plate, the plate exposed to abacteriophage or polypeptide of the invention, and subsequent growth ofthe colonies evaluated relative to control plates. Decreased size ofcolonies, or decreased total numbers of colonies, indicates abacteriophage and/or polypeptide with antibacterial activity against thetested sample and/or cultured species or strain.

The present invention is also directed to pharmaceutical compositionscomprising or consisting of a bacteriophage having a genome comprisingor consisting of the nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, or SEQ IDNO:760. In certain embodiments, the pharmaceutical composition of theinvention comprises a bacteriophage having the genome comprising orconsisting of the nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, or SEQ IDNO:760 in addition to one or more other bacteriophages. The one or moreother bacteriophages may be one or more bacteriophages of the invention(e.g., having a genome comprising or consisting of a nucleic acidsequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 or SEQ ID NO:7, or SEQ ID NO:760), one or more strainsthereof, or may be one or more bacteriophages known in the art. Further,the one or more bacteriophages in the pharmaceutical composition of theinvention may target the same or different species or strains ofbacteria. In certain embodiments, the pharmaceutical compositionscomprising one or more bacteriophages of the invention further compriseone or more polypeptides of the invention and/or other phage products asdescribed herein or known in the art.

In certain embodiments, the invention provides pharmaceuticalcompositions comprising polypeptides, or active fragments thereof, inparticular those having anti-microbial and/or antibacterial activity,isolated from bacteriophage having a genome comprising or consisting ofthe nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6 SEQ ID NO:7, and/or SEQ ID NO:760. Inspecific embodiments, the pharmaceutical compositions of the inventioncomprise one or more polypeptides having an amino acid sequence of SEQID NO:61, SEQ ID NO:63, SEQ ID NO:68, SEQ ID NO:184, SEQ ID NO:202, SEQID NO:203, SEQ ID NO:204, SEQ ID NO:214, SEQ ID NO:435, SEQ ID NO:438,SEQ ID NO:440, SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQ IDNO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534,SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ IDNO:539, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:575, SEQ ID NO:632, SEQID NO:633, SEQ ID NO:641, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702,SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:712, SEQ ID NO:755, SEQ IDNO:756, SEQ ID NO:757, SEQ ID NO:758, SEQ ID NO:759 or SEQ IDNOS:761-816. In other embodiments, the pharmaceutical compositions ofthe invention comprise a polypeptide that is a variant, derivative orfragment of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:68, SEQ ID NO:184, SEQID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:214, SEQ ID NO:435,SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:446, SEQ ID NO:447, SEQ IDNO:448, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533,SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ IDNO:538, SEQ ID NO:539, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:575, SEQID NO:632, SEQ ID NO:633, SEQ ID NO:641, SEQ ID NO:700, SEQ ID NO:701,SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ ID NO:712, SEQ IDNO:755, SEQ ID NO:756, SEQ ID NO:757, SEQ ID NO:758, SEQ ID NO:759, orSEQ ID NOS:761-816 wherein the variant, derivative or fragment retains abiologic function of the polypeptide from which it is derived, e.g.,antimicrobial or antibacterial activity (e.g., lytic killing activity),preferably against one or more strains of A. baumannii, E. faecalis, E.faecium, P. aeruginosa and/or S. aureus.

The pharmaceutical compositions of the invention may additionallycomprise a pharmaceutically acceptable carrier, excipient, orstabilizer. In certain embodiments, the pharmaceutical compositions ofthe invention are antibiotic compositions (in that they exhibitantibacterial activity) or therapeutic compositions for the treatment,prevention, and/or amelioration of symptoms of a disease or disorderassociated with infection by bacteria in a subject in need thereof. Inspecific embodiments, the pharmaceutical compositions of the inventionare antibacterial compositions or therapeutic compositions for thetreatment, prevention, and/or amelioration of symptoms of a disease ordisorder associated with infection by A. baumannii, E. faecalis, E.faecium, P. aeruginosa and/or S. aureus. In certain embodiments, thesubject receiving a pharmaceutical composition of the invention is amammal (e.g., bovine, ovine, caprine, equid, primate (e.g., human),rodent, lagomorph or avian (e.g., chicken, duck, goose)).

The present invention provides for methods for the treatment orprevention of bacterial infection comprising administering to a subjectin need thereof a pharmaceutical composition comprising one or morebacteriophages or phage products (e.g., an isolated bacteriophagepolypeptide or active fragment, variant or derivative thereof),optionally in addition to one or more other bacteriophages or otherphage products, as described herein. In the context of the presentinvention, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is toeliminate, lessen, decrease the severity of, slow the progression of ordelay or prevent the symptoms or underlying cause (e.g., bacterialinfection) associated with the pathological condition or disorder. Thepharmaceutical compositions of the present invention may be used in thetreatment or management of infections associated with any bacterialinfection, including, but not limited to A. baumanni, S. aureus, S.epidermidis, S. auricularis, S. capitis, S. haemolyticus, S. hominis, S.saprophyticus, S. simulans, S. xylosis, M. luteus, B. subtilis, B.pumilus, E. faecalis, E. hirae, E. faecium, E. avium, P. aeruginosa andcombinations thereof. In certain embodiments, the pharmaceuticalcompositions may be used to treat conditions or disorders associatedwith bacterial infections including, but not limited to, post-operativeendophtalmitis, endocarditis, infections of the central nervous system,pneumonia, osteomylelitis, wound infections (e.g., diabetic footulcers), mastitis, septicemia, food poisoning and meningitis and/orother conditions associated with nosocomial bacterial infections.

In certain embodiments, the invention provides for the use of abacteriophage or an isolated phage product (e.g., an isolated phagepolypeptide or active fragment, variant or derivative thereof) as asingle agent therapy. In other embodiments, the invention provides forthe use of a bacteriophage, or phage product (e.g., an isolated phagepolypeptide or active fragment, variant or derivative thereof), incombination with a standard or experimental treatment for bacterialinfection. Such combination therapy may enhance the efficacy of thestandard or experimental treatment. Examples of therapeutic agents thatare particularly useful in combination with a polypeptide of theinvention are anti-inflammatory agents, standard chemotherapeuticantibiotic agents (e.g., penicillin, synthetic penicillins, bacitracin,methicillin, cephalosporin, polymyxin, cefaclor, Cefadroxil, cefamandolenafate, cefazolin, cefixime, cefmetazole, cefonioid, cefoperazone,ceforanide, cefotanme, cefotaxime, cefotetan, cefoxitin, cefpodoximeproxetil, ceftazidime, ceftizoxime, ceftriaxone, cefriaxone moxalactam,cefuroxime, cephalexin, cephalosporin C, cephalosporin C sodium salt,cephalothin, cephalothin sodium salt, cephapirin, cephradine,cefuroximeaxetil, dihydratecephalothin, moxalactam, loracarbef mafateand chelating agents), local anesthetic agents, and/or corticosteroids.In yet another embodiment, the compositions of the present invention maybe combined with one or more bacteriophages or phage products known inthe art. The combination therapies encompassed by the invention may beformulated into a single pharmaceutical composition or may beadministered in separate compositions, but as part of an overalltreatment regimen.

The pharmaceutical compositions of the invention may be administered byany method known in the art suitable for administration of anantibacterial compound (e.g., via oral or parenteral (e.g., inhalation,intramuscular, intravenous, or epidermal)) delivery. In preferredembodiments, the pharmaceutical compositions of the invention areadministered topically, e.g., in a topical formulation. The compositionsof the invention may be used topically to treat and/or prevent commonnosocomial infections, such as infections at surgical incision sites orassociated with catheters or drains. In other embodiments, thecompositions of the invention are use to treat bacterial infections ofthe skin or upper dermal layers (e.g., infections of diabetic ulcers ofthe foot).

The pharmaceutical compositions of the present invention may also beused for traditionally non-therapeutic uses such as antibacterial agentsin cosmetics, or in sprays or solutions for use on solid surfaces toprevent the colonization of bacteria (i.e., as disinfectants).

The present invention is also directed to methods for screening peptidesfor antibacterial activity. In one embodiment the method comprisesscreening contiguous amino acid sequences of at least 6, 10, 15, 20 or25 residues in length that are encoded by the open reading frames of thenucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:760 forantibacterial activity, said antibacterial activity measured by thepeptides ability to inhibit bacterial growth in agar or liquid culture.

4.1 DEFINITIONS

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of aprotein. In a specific embodiment, the fragment is a functional fragmentin that it retains at least one function of the protein from which it isisolated (e.g., antimicrobial or antibacterial activity (e.g., lyticcell killing)).

As used herein the terms “active bacteriophage products” and“bacteriophage products” refer to polypeptides, or fragments, variantsor derivatives thereof, isolated from a bacteriophage of the invention,which polypeptide, or fragment, variant or derivative thereof, exhibitsa biological function or activity associated with the bacteriophage fromwhich it was isolated or derived (e.g., antimicrobial or antibacterialactivity (e.g., lytic cell killing)).

As used herein, the term “isolated” in the context of a peptide,polypeptide, or fusion protein or refers to a peptide, polypeptide orfusion protein that is substantially free of cellular material orcontaminating proteins from the cell or tissue source from which it isderived, or substantially free of chemical precursors or other chemicalswhen chemically synthesized. The language “substantially free ofcellular material” includes preparations of a peptide, polypeptide orfusion protein in which the peptide, polypeptide or fusion protein isseparated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, a peptide, polypeptide orfusion protein that is substantially free of cellular material includespreparations of a peptide, polypeptide or fusion protein having lessthan about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein(also referred to herein as a “contaminating protein”). When thepeptide, polypeptide or fusion protein is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, 10%, or 5% of the volume of theprotein preparation. When the peptide, polypeptide or fusion protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the peptide, polypeptide or fusion protein. Accordinglysuch preparations of a peptide, polypeptide, fusion protein or antibodyhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the peptide, polypeptide or fusionprotein of interest.

As used herein, the term “isolated” in the context of nucleic acidmolecules refers to a nucleic acid molecule which is separated fromother nucleic acid molecules which are present in the natural source ofthe first nucleic acid molecule. Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized and may be free of cDNA or othergenomic DNA molecules, e.g., has been isolated from other clones in anucleic acid library.

The term “purified” means that the peptide, polypeptide, fusion proteinor nucleic acid molecule has been measurably increased in concentrationby any purification process, including but not limited to, columnchromatography, HPLC, precipitation, electrophoresis, etc., therebypartially, substantially, or completely removing impurities such asprecursors or other chemicals involved in preparing the peptide,polypeptide, fusion protein or nucleic acid molecule. One of skill inthe art will appreciate the amount of purification necessary for a givenuse. For example, isolated protein meant for use in therapeuticcompositions intended for administration to humans ordinarily must be ofhigh purity in accordance with regulatory standards and goodmanufacturing processes.

As used herein, the term “derivative” in the context of polypeptidesrefers to a polypeptide that comprises an amino acid sequence which hasbeen altered by the introduction of amino acid residue substitutions,deletions or additions. The term “derivative” as used herein also refersto a polypeptide that has been modified, i.e., by the covalentattachment of any type of molecule to the polypeptide. For example, butnot by way of limitation, a polypeptide may be modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Aderivative polypeptide may be produced by chemical modifications usingtechniques known to those of skill in the art, including, but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Further, a derivativepolypeptide may contain one or more non-classical amino acids. Apolypeptide derivative possesses a similar or identical function as thepolypeptide from which it was derived. The term “derived” as used inreference to a polypeptide “derived” from an organism may also refer toisolation of a polypeptide directly from said organism (e.g. bacterialcells or phage).

As used herein, the term “host cell” refers to the particular subjectcell transfected with a nucleic acid molecule and the progeny orpotential progeny of such a cell that contain the nucleic acid moleculeor chromosomally integrated version thereof. Progeny of such a cell maynot be identical to the parent cell transfected with the nucleic acidmolecule due to mutations or environmental influences that may occur insucceeding generations or integration of the nucleic acid molecule intothe host cell genome. For the expression of bacteriophage proteins andpolypeptides, the host cell is preferably not of the same species orstrain from which the bacteriophage was isolated or cultured.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agent. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disease ordisorder. A first prophylactic or therapeutic agent can be administeredprior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second prophylactic or therapeutic agent (differentfrom the first prophylactic or therapeutic agent) to a subject with adisease or disorder.

As used herein, the terms “nucleic acids” and “nucleotide sequences”include single-stranded and double-stranded DNA and/or RNA molecules, orcombinations thereof. As used herein, the term “encoded by the nucleicacid” refers to an amino acid sequence that results from the translationof the forward, reverse, complementary or reverse-complementary sequenceof the referenced nucleic acid sequence using the standard genetic code(i.e., standard codon triplets) as well known in the art.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to bacteriophages and/or polypeptides of the invention, which canbe used in the prevention, treatment, management or amelioration of oneor more symptoms associated with infection by a bacterium.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to bacteriophages and/or polypeptides of the invention that can beused in the prevention, treatment, management or amelioration of one ormore symptoms of a disease or disorder, in particular, a disease ordisorder associated with a bacterial infection.

As used herein, the term “therapeutically effective amount” refers tothat amount of a therapeutic agent sufficient to result in ameliorationof one or more symptoms of a disease or disorder, in particular, adisease or disorder associated with a bacterial infection.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe amelioration of one or more symptoms associated with a bacterialinfection that results from the administration of one or morebacteriophages and/or polypeptides of the invention. As noted above,“treatment” and related terms refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is toeliminate, lessen, decrease the severity of, slow the progression of, ordelay or prevent the symptoms or underlying cause (e.g., bacterialinfection) associated with the pathological condition or disorder.

As used herein, the terms “antibacterial activity” and “antimicrobialactivity” with reference to a bacteriophage, isolated bacteriophageprotein (or variant, derivative or fragment thereof), or bacteriophageproduct, are used interchangeably to refer to the ability to kill and/orinhibit the growth or reproduction of a microorganism, in particular,the bacteria of the species or strain that the bacteriophage infects. Incertain embodiments, antibacterial or antimicrobial activity is assessedby culturing bacteria (e.g., Gram-positive bacteria (e.g., E. faecalis,E. faecium, S. aureus), Gram-negative bacteria (e.g., A. baumannii, P.aeruginosa) or bacteria not classified as either Gram-positive orGram-negative) according to standard techniques (e.g., in liquid cultureor on agar plates), contacting the culture with a bacteriophage orpolypeptide of the invention and monitoring cell growth after saidcontacting. For example, in a liquid culture, the bacteria may be grownto an optical density (“OD”) representative of a mid-point inexponential growth of the culture; the culture is exposed to one or moreconcentrations of one or more bacteriophages or polypeptides of theinvention and the OD is monitored relative to a control culture.Decreased OD relative to a control culture is representative of abacteriophage or polypeptide exhibiting antibacterial activity (e.g.,exhibits lytic killing activity). Similarly, bacterial colonies can beallowed to form on an agar plate, the plate exposed to a bacteriophageor polypeptide of the invention, and subsequent growth of the coloniesevaluated related to control plates. Decreased size of colonies, ordecreased total numbers of colonies, indicate a bacteriophage orpolypeptide with antibacterial activity.

As used herein, a “CHAP domain” refers to a conserved amidase domainfound in several phage-encoded peptidoglycan hydrolases and stands forfor “cysteine, histidine-dependent amidohydrolases/peptidases.” See,e.g., Rigden D, et. al., Trends Biochem Sci. 2003 May 28(5): 230-4. Itis found in a superfamily of amidases, including GSP amidase andpeptidoglycan hydrolases. The family includes at least two differenttypes of peptidoglycan cleavage activities: L-muramoyl-L-alanine amidaseand D-alanyl-glycyl endopeptidase activity. CHAP domains generallycontain conserved cysteine and histidine residues and hydrolyzeγ-glutamyl-containing substrates. These cysteine residues are believedto be essential for the activity of several of these amidases, and theirthiol groups appear to function as the nucleophiles in the catalyticmechanisms of all enzymes containing this domain. CHAP domains are oftenfound in association with other domains that cleave peptidoglycan, e.g.,acting in a cooperative manner to cleave specialized substrates. Seealso, Bateman A, et al., Trends Biochem Sci. 2003 May 28(5): 234-7.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic of the organization of the F168/08 genome, comprisingthe nucleic acid sequence of SEQ ID NO:1. The open reading frames(“ORFs”) predicted in the genome are represented by arrows and numberedin black. The direction of an arrow indicates the direction oftranscription. Color coding: Black—ORFs for which products a functionalassignment could be made based on the known functions of homologousproteins (ORFs encoding products exhibiting homology to the same orsimilar proteins are indicated using the same number and differentiatedby lowercase letter); Gray—ORFs coding for products that are similar toproteins of unknown function; White or Empty—ORFs coding for proteinsthat share no significant homology with proteins in available databases.Functionally assigned ORFs are also listed in the figure. Theinformation in the figure is also included in tabular form in FIG. 2.

FIGS. 2A-X: Features of the bacteriophage F168/08 genome, including geneproducts and assignment of putative functions. The figure includes alisting of the ORFs of the genome and provides for each ORF (i) itsposition within the genome, (ii) the encoded amino acid sequence, (iii)a listing of homologous proteins and conserved domains within itsencoded polypeptide and (iv) an assignment of putative function. ORFs1-116 listed in FIG. 2 encode the amino acid sequences of SEQ IDNO:8-130, respectively.

FIGS. 3A-B: The host range of F168/08 as determined by the spot test in105 Enterococcus faecalis (EFS) (3A) and 56 Enterococcus faecium (EFM)(3B) strains isolated from clinical samples. Each spot contained 5 μlbacteriophage suspension with indicated titers (prepared from CsClpurified lysate). Sensitivity of each strain to the phage was evaluatedbased on a relative scale ranging from turbid (+) to clear (++++) lysishalos. Resistance to phage infection is indicated as (−).

FIG. 4: Schematic of the organization of the F170/08 genome, comprisingthe nucleic acid sequence of SEQ ID NO:2. The ORFs predicted in thegenome are represented by arrows and numbered in black. The direction ofan arrow indicates the direction of transcription. Color coding:Black—ORFs for which products a functional assignment could be madebased on the known functions of homologous proteins (ORFs encodingproducts exhibiting homology to the same or similar proteins areindicated using the same number and differentiated by lowercase letter);Gray—ORFs coding for products that are similar to proteins of unknownfunction; White or Empty—ORFs coding for proteins that share nosignificant homology with proteins in available databases. Functionallyassigned ORFs are also listed in the figure. The information in thefigure is also included in tabular form in FIG. 5.

FIGS. 5A-AU: Features of the bacteriophage F170/08 genome, includinggene products and assignment of putative functions. The figure includesa listing of the ORFs of the genome and provides for each ORF (i) itsposition within the genome, (ii) the encoded amino acid sequence, (iii)a listing of homologous proteins and conserved domains within itsencoded polypeptide and (iv) an assignment of putative function. ORFs1-213 listed in FIG. 5 encode the amino acid sequences of SEQ IDNO:131-343, respectively.

FIGS. 6A-B: The host range of F170/08 as determined by the spot test in105 Enterococcus faecalis (EFS) (6A) and 56 Enterococcus faecium (EFM)(6B) strains isolated from clinical samples. Each spot contained 5 μlbacteriophage suspension with indicated titers (prepared from CsClpurified lysate). Sensitivity of each strain to the phage was evaluatedbased on a relative scale ranging from turbid (+) to clear (++++) lysishalos. Resistance to phage infection is indicated as (−).

FIG. 7: Schematic of the organization of the F770/05 genome, comprisingthe nucleic acid sequence of SEQ ID NO:3. The ORFs predicted in thegenome are represented by arrows and numbered in black. The direction ofan arrow indicates the direction of transcription. Color coding:Black—ORFs for which products a functional assignment could be madebased on the known functions of homologous proteins (ORFs encodingproducts exhibiting homology to the same or similar proteins areindicated using the same number and differentiated by lowercase letter);Gray—ORFs coding for products that are similar to proteins of unknownfunction; White or Empty—ORFs coding for proteins that share nosignificant homology with proteins in available databases. Functionallyassigned ORFs are also listed in the figure. The information in thefigure is also included in tabular form in FIG. 8.

FIGS. 8A-AE: Features of the bacteriophage F770/05 genome, includinggene products and assignment of putative functions. The figure includesa listing of the ORFs of the genome and provides for each ORF (i) itsposition within the genome, (ii) the encoded amino acid sequence, (iii)a listing of homologous proteins and conserved domains within itsencoded polypeptide and (iv) an assignment of putative function. ORFs1-95 listed in FIG. 8 encode the amino acid sequences of SEQ IDNO:344-438, respectively.

FIG. 9: The host range of F770/05 as determined by the spot test in 100Pseudomonas aeruginosa (PSA) strains isolated from clinical samples.Each spot contained 5 μl bacteriophage suspension with indicated titers(prepared from CsCl purified lysate). Sensitivity of each strain to thephage was evaluated based on a relative scale ranging from turbid (+) toclear (++++) lysis halos. Resistance to phage infection is indicated as(−).

FIG. 10: Schematic of the organization of the F197/08 genome, comprisingthe nucleic acid sequence of SEQ ID NO:4. The ORFs predicted in thegenome are represented by arrows and numbered in black. The direction ofan arrow indicates the direction of transcription. Color coding:Black—ORFs for which products a functional assignment could be madebased on the known functions of homologous proteins (ORFs encodingproducts exhibiting homology to the same or similar proteins areindicated using the same number and differentiated by lowercase letter);Gray—ORFs coding for products that are similar to proteins of unknownfunction; White or Empty—ORFs coding for proteins that share nosignificant homology with proteins in available databases. Functionallyassigned ORFs are also listed in the figure. The information in thefigure is also included in tabular form in FIG. 11.

FIGS. 11A-AA: Features of the bacteriophage F197/08 genome, includinggene products and assignment of putative functions. The figure includesa listing of the ORFs of the genome and provides for each ORF (i) itsposition within the genome, (ii) the encoded amino acid sequence, (iii)a listing of homologous proteins and conserved domains within itsencoded polypeptide and (iv) an assignment of putative function. ORFs1-66 listed in FIG. 11 encode the amino acid sequences of SEQ IDNO:439-553, respectively.

FIG. 12: The host range of F197/08 as determined by the spot test in 100Staphylococcus aureus (STA) strains isolated from clinical samples. Eachspot contained 5 μl bacteriophage suspension with indicated titers(prepared from CsCl purified lysate). Sensitivity of each strain to thephage was evaluated based on a relative scale ranging from turbid (+) toclear (++++) lysis halos. Resistance to phage infection is indicated as(−).

FIG. 13: Schematic of the organization of the F86/06 genome, comprisingthe nucleic acid sequence of SEQ ID NO:5. The ORFs predicted in thegenome are represented by arrows and numbered in black. The direction ofan arrow indicates the direction of transcription. Color coding:Black—ORFs for which products a functional assignment could be madebased on the known functions of homologous proteins (ORFs encodingproducts exhibiting homology to the same or similar proteins areindicated using the same number and differentiated by lowercase letter);Gray—ORFs coding for products that are similar to proteins of unknownfunction; White or Empty—ORFs coding for proteins that share nosignificant homology with proteins in available databases. Functionallyassigned ORFs are also listed in the figure. The information in thefigure is also included in tabular form in FIG. 14.

FIGS. 14A-U: Features of the bacteriophage F86/06 genome, including geneproducts and assignment of putative functions. The figure includes alisting of the ORFs of the genome and provides for each ORF (i) itsposition within the genome, (ii) the encoded amino acid sequence, (iii)a listing of homologous proteins and conserved domains within itsencoded polypeptide and (iv) an assignment of putative function. ORFs1-63 listed in FIG. 14 encode the amino acid sequences of SEQ IDNO:554-616, respectively.

FIG. 15: The host range of F86/06 as determined by the spot test in 100Staphylococcus aureus (STA) strains isolated from clinical samples. Eachspot contained 5 μl bacteriophage suspension with indicated titers(prepared from CsCl purified lysate). Sensitivity of each strain to thephage was evaluated based on a relative scale ranging from turbid (+) toclear (++++) lysis halos. Resistance to phage infection is indicated as(−).

FIG. 16: Schematic of the organization of the F87s/06 genome, comprisingthe nucleic acid sequence of SEQ ID NO:6. The ORFs predicted in thegenome are represented by arrows and numbered in black. The direction ofan arrow indicates the direction of transcription. Color coding:Black—ORFs for which products a functional assignment could be madebased on the known functions of homologous proteins (ORFs encodingproducts exhibiting homology to the same or similar proteins areindicated using the same number and differentiated by lowercase letter);Gray—ORFs coding for products that are similar to proteins of unknownfunction; White or Empty—ORFs coding for proteins that share nosignificant homology with proteins in available databases. Functionallyassigned ORFs are also listed in the figure. The information in thefigure is also included in tabular form in FIG. 17.

FIGS. 17A-V: Features of the bacteriophage F87s/06 genome, includinggene products and assignment of putative functions. The figure includesa listing of the ORFs of the genome and provides for each ORF (i) itsposition within the genome, (ii) the encoded amino acid sequence, (iii)a listing of homologous proteins and conserved domains within itsencoded polypeptide and (iv) an assignment of putative function. ORFs1-61 listed in FIG. 17 encode the amino acid sequences of SEQ IDNO:617-681, respectively.

FIG. 18: The host range of F87s/06 as determined by the spot test in 100Staphylococcus aureus (STA) strains isolated from clinical samples. Eachspot contained 5 μl bacteriophage suspension with indicated titers(prepared from CsCl purified lysate). Sensitivity of each strain to thephage was evaluated based on a relative scale ranging from turbid (+) toclear (++++) lysis halos. Resistance to phage infection is indicated as(−).

FIG. 19: Schematic of the organization of the F91a/06 genome, comprisingthe nucleic acid sequence of SEQ ID NO:7. The ORFs predicted in thegenome are represented by arrows and numbered in black. The direction ofan arrow indicates the direction of transcription. Color coding:Black—ORFs for which products a functional assignment could be madebased on the known functions of homologous proteins (ORFs encodingproducts exhibiting homology to the same or similar proteins areindicated using the same number and differentiated by lowercase letter);Gray—ORFs coding for products that are similar to proteins of unknownfunction; White or Empty—ORFs coding for proteins that share nosignificant homology with proteins in available databases. Functionallyassigned ORFs are also listed in the figure. The information in thefigure is also included in tabular form in FIG. 20.

FIGS. 20A-U: Features of the bacteriophage F91a/06 genome, includinggene products and assignment of putative functions. The figure includesa listing of the ORFs of the genome and provides for each ORF (i) itsposition within the genome, (ii) the encoded amino acid sequence, (iii)a listing of homologous proteins and conserved domains within itsencoded polypeptide and (iv) an assignment of putative function. ORFs1-64 listed in FIG. 20 encode the amino acid sequences of SEQ IDNO:682-754, respectively.

FIG. 21: The host range of F91a/06 as determined by the spot test in 100Staphylococcus aureus (STA) strains isolated from clinical samples. Eachspot contained 5 μl bacteriophage suspension with indicated titers(prepared from CsCl purified lysate). Sensitivity of each strain to thephage was evaluated based on a relative scale ranging from turbid (+) toclear (++++) lysis halos. Resistance to phage infection is indicated as(−).

FIG. 22: Schematic organization of the F1245/05 genome, comprising thenucleic acid sequence of SEQ ID NO:760. The ORFs predicted in the 43kbgenome are represented by arrows and numbered in black. For the ORFsfunctionally assigned, the number is substituted by the predictedfunction. The direction of an arrow indicates the direction oftranscription. Color coding: Black—ORFs for whose products a functionalassignment could be made based on homologous proteins; Gray—ORFs codingfor products that are similar to proteins of unknown function;Striped—ORFs with an assigned function based on its relative genomeposition and on the presence of putative transmembrane domains in theencoded product; Empty arrows—ORFs coding for proteins that share nosignificant homology with proteins in databases.

FIGS. 23A-O: Features of bacteriophage F1245/05 genome, including geneproducts and assignment of putative functions are provided.

FIGS. 24A-E: The host range of F1245/05 as determined by the spot testin 100 Acinetobacter baumannii strains isolated from clinical samples isprovided. Each spot contained 5 μl bacteriophage suspensions withindicated titers (prepared from CsCI purified lysate). Sensitivity tothe phage is presented as a scale ranging from turbid (+) to clear(++++) lysis halos. Resistance to phage infection is indicated as (−).

6. DETAILED DESCRIPTION

The present invention is directed to isolated bacteriophages, and theirisolated polypeptide products, having antibacterial activity against oneor more species or strains of the nosocomial pathogens A. baumannii, E.faecalis, E. faecium, P. aeruginosa and S. aureus. In one embodiment,isolated bacteriophages or polypeptides are provided that exhibitantimicrobial and/or antibacterial activity against methicillinresistant strains of S. aureus (MRSA). In addition, the bacteriophagesand polypeptides of the invention may exhibit antibacterial orantimicrobial activity against one or more species or strains ofpathogenic bacteria including, but not limited to, S. epidermidis, S.auricularis, S. capitis, S. haemolyticus, S. hominis, S. saprophyticus,S. simulans, S. xylosis, Micrococcus luteus, Bacilus subtilis, B.pumilus, E. hirae and E. avium.

In one embodiment, the invention provides a bacteriophage having agenome comprising or consisting of the nucleic acid sequence of SEQ IDNO:1. A specific example in accordance with this embodiment is theisolated bacteriophage F168/08, which targets a number of strains of E.faecalis and E. faecium. Open reading frames (ORFs) in the F168/08genome, the amino acid sequences encoded by the ORFs and the assignmentof putative functions of the encoded amino acid sequences (i.e., encodedproteins and/or polypeptides) are provided in FIG. 2 (also providing theamino acid sequences SEQ ID NOS:8-130).

Enterococci are gram-positive, spherical bacteria that form colonies ingroups or chains. They are found as part of the digestive tract flora inmany mammals, including humans. Enterococcus infections account for 12%of all nosocomial infections. An Enterococcus infection can causecomplicated abdominal infections, skin and skin structure infections,urinary tract infections and infections of the blood stream, which canbe difficult to treat, particularly in cases where the strain involvedhas developed resistance to several antibiotics. In such instances,infection can be life threatening, especially where the patient isalready immunodeficient.

Enterococcus faecalis accounts for the majority of Enterococciinfections and is a Gram-positive commensal bacterium inhabiting thegastrointestinal tracts of humans and other mammals. It is non-motileand facultatively anaerobic. E. faecalis can cause endocarditis, as wellas bladder, prostate, and epididymal infections, including lifethreatening infections in humans, especially in the nosocomialenvironment. E. faecalis is resistant to many commonly usedantimicrobial agents (such as, e.g., aminoglycosides, aztreonam,cephalosporins, clindamycin, the semi-synthetic penicillins nafcillinand oxacillin, trimethoprim-sulfamethoxazole, and the like).

Enterococcus faecium is known to have a resistance to several types ofantibiotics including quinolones and aminoglycosides.Vancomycin-resistant strains of E. faecium are also known. Resistance toseveral antibiotics and tolerance for adverse conditions makes E.faecium a major concern for the medical community, which has dubbed thismicrobe a “supergerm”. In another embodiment, the invention provides abacteriophage having a genome comprising or consisting of the nucleicacid sequence of SEQ ID NO:2. A specific example in accordance with thisembodiment is the isolated bacteriophage F170/08, which targets a numberof strains of E. faecalis and E. faecium. Open reading frames (ORFs) inthe F178/08 genome, the amino acid sequences encoded by the ORFs and theassignment of putative functions of the encoded amino acid sequences(i.e., encoded proteins and/or polypeptides) are provided in FIG. 5(also providing the amino acid sequences SEQ ID NOS:131-343).

In still another embodiment, the invention provides a bacteriophagehaving a genome comprising or consisting of the nucleic acid sequence ofSEQ ID NO:3. A specific example in accordance with this embodiment isthe isolated bacteriophage F770/05, which targets a number of strains ofP. aeruginosa. Open reading frames (ORFs) in the F770/05 genome, theamino acid sequences encoded by the ORFs and the assignment of putativefunctions of the encoded amino acid sequences (i.e., encoded proteinsand/or polypeptides) are provided in FIG. 8 (also providing the aminoacid sequences SEQ ID NOS:344-438).

Pseudomonas aeruginosa is a common Gram-negative rod-shaped bacteriumfound in soil, water, skin flora and most man-made environments. Itthrives not only in normal atmospheres, but also with little oxygen as afacultative anaerobe, and can infect damaged tissues or immunocomromisedinidviduals. When such colonisations occur in critical body organs suchas the lungs, the urinary tract, and kidneys, the results can be fatal.Because it thrives on surfaces, this bacterium is also found on and inmedical equipment including catheters, causing cross infections inhospitals and clinics. P. aeruginosa is one of the most relevantopportunistic, nosocomial pathogens, and it has been estimated that onein ten hosptical-acquired infections are from Pseudomonas. P. aeruginosais also the most common cause of burn injury infections and the mostfrequent colonizer of medical devices, such as catheters.

In yet another embodiment, the invention provides a bacteriophage havinga genome comprising or consisting of the nucleic acid sequence of SEQ IDNO:4. A specific example in accordance with this embodiment is theisolated bacteriophage F197/08, which targets a number of strains of S.aureus. Open reading frames (ORFs) in the F197/08 genome, the amino acidsequences encoded by the ORFs and the assignment of putative functionsof the encoded amino acid sequences (i.e., encoded proteins and/orpolypeptides) are provided in FIG. 11 (also providing the amino acidsequences SEQ ID NOS:439-553).

In yet another embodiment, the invention provides a bacteriophage havinga genome comprising or consisting of the nucleic acid sequence of SEQ IDNO:5. A specific example in accordance with this embodiment is theisolated bacteriophage F86/06, which targets a number of strains of S.aureus. Open reading frames (ORFs) in the F86/06 genome, the amino acidsequences encoded by the ORFs and the assignment of putative functionsof the encoded amino acid sequences (i.e., encoded proteins and/orpolypeptides) are provided in FIG. 14 (also providing the amino acidsequences SEQ ID NOS:554-616).

In yet another embodiment, the invention provides a bacteriophage havinga genome comprising or consisting of the nucleic acid sequence of SEQ IDNO:6. A specific example in accordance with this embodiment is theisolated bacteriophage F87s/06, which targets a number of strains of S.aureus. Open reading frames (ORFs) in the F87s/06 genome, the amino acidsequences encoded by the ORFs and the assignment of putative functionsof the encoded amino acid sequences (i.e., encoded proteins and/orpolypeptides) are provided in FIG. 17 (also providing the amino acidsequences SEQ ID NOS:617-681).

In still another embodiment, the invention provides a bacteriophagehaving a genome comprising or consisting of the nucleic acid sequence ofSEQ ID NO:7. A specific example in accordance with this embodiment isthe isolated bacteriophage F91a/06, which targets a number of strains ofS. aureus. Open reading frames (ORFs) in the F91a/06 genome, the aminoacid sequences encoded by the ORFs and the assignment of putativefunctions of the encoded amino acid sequences (i.e., encoded proteinsand/or polypeptides) are provided in FIG. 20 (also providing the aminoacid sequences SEQ ID NOS:682-754).

S. aureus is a spherical, facultatively-anaerobic, Gram-positivebacterium, often part of the skin flora found in the nose and on skin.S. aureus can cause a range of illnesses from minor skin infections,such as pimples, to gastroenteritis, to life-threatening diseases suchas pneumonia, meningitis, osteomyelitis, endocarditis, toxic shocksyndrome (TSS), bacteremia, and sepsis. It is one of the five mostcommon causes of nosocomial infections, and often the cause ofpostsurgical wound infections. Today, S. aureus has become resistant tomany commonly used antibiotics, and it has been estimated that only 2%of all S. aureus isolates are sensitive to penicillin. Second-generationpenicillins, such s methicillin, oxacillin, cloxacillin andflucloxacillin, were developed to treat penicillin-resistant S. aureus.Methicillin was the first antibiotic in this class to be used, but onlytwo years later, the first case of methicillin-resistant S. aureus(MRSA) was reported. Since the 1990s, there has been an explosion inMRSA prevalence in hospitals, where it is now considered an endemic.

In still another embodiment, the invention provides a bacteriophagehaving a genome comprising or consisting of the nucleic acid sequence ofSEQ ID NO:760. A specific example in accordance with this embodiment isthe isolated bacteriophage F1245/05, which targets a number of strainsof A. baumannii. Open reading frames (ORFs) in the F1245/05 genome, theamino acid sequences encoded by the ORFs and the assignment of putativefunctions of the encoded amino acid sequences (i.e., encoded proteinsand/or polypeptides) are provided in FIG. 23 (also providing the aminoacid sequences SEQ ID NOS:761-816).

Acinetobacter baumannii is a species of bacteria that causes a number ofsevere clinical infections, particularly in individuals with compromisedimmune systems. Acinetobacter baumannii is a pleomorphic aerobicgram-negative bacillus that is commonly isolated from the hospitalenvironment and from hospitalized patients. The bacterium often entersthe body open wounds, catheters, or breathing tubes. Acinetobacterbaumannii usually colonizes aquatic environments and is often culturedfrom hospitalized patients' sputum or respiratory secretions, wounds,and urine. In a hospital setting, Acinetobacter baumannii commonlycolonizes irrigating solutions and intravenous solutions. It is alsoknown to be resistant to multiple antibiotics and the number ofnosocomial infections caused by A. baumanni has increased in recentyears.

In certain embodiments, the bacteriophage of the invention comprises orconsists of a genome having a sequence identity of at least 85%, 90%,95%, 96%, 97%, 98% or at least 99% with the nucleic acid sequence of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, or SEQ ID NO:760, which bacteriophage exhibits atleast one biological, e.g., antimicrobial or antibacterial activity(e.g., lytic killing activity), of one or more of bacteriophage F168/08,F170/08, F770/05, F197/08, F86/06, F87s/06, F91a/06 and F1245/05.Alternatively or in addition, the bacteriophage of the invention mayhave a genome comprising a functional fragment of the nucleic acidsequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:760, including thesequences of any of the open reading frames described in FIGS. 2, 5, 8,11, 14, 17, 20, and 23.

The invention also provides for isolated bacteria infected with one ormore of the bacteriophages of the invention. In certain embodiments, theinvention provides isolated E. faecalis or E. faecium infected with abacteriophage having a genome comprising or consisting of the nucleicacid sequence of SEQ ID NO:1 and/or SEQ ID NO:2. In other embodiments,the invention provides isolated P. aeruginosa infected with abacteriophage having a genome comprising or consisting of the nucleicacid sequence of SEQ ID NO:3. In still other embodiments, the inventionprovides isolated S. aureus infected with a bacteriophage having agenome comprising or consisting of the nucleic acid sequence of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6 and/or SEQ ID NO:7. In yet still otherembodiments, the invention provides isolated A. baumannii infected witha bacteriophage having a genome comprising or consisting of the nucleicacid sequence of SEQ ID NO:760.

The invention provides for methods of production and isolation of abacteriophage having a genome comprising or consisting of the nucleicacid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:760. In certainembodiments, the invention provides for a method of producing and/orisolating a bacteriophage having a genome that comprises or consists ofthe nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2 comprising (i)obtaining a culture of E. faecalis or E. faecium, (ii) infecting it withthe bacteriophage having a genome comprising or consisting of thenucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2; (iii) culturinguntil significant lysis of the culture is observed and (iv) isolatingfrom the culture the bacteriophage. In other embodiments, the inventionprovides for a method of producing and/or isolating a bacteriophagehaving a genome that comprises or consists of the nucleic acid sequenceof SEQ ID NO:3 comprising (i) obtaining a culture of P. aeruginosa, (ii)infecting it with the bacteriophage having a genome comprising orconsisting of the nucleic acid sequence of SEQ ID NO:3; (iii) culturinguntil significant lysis of the culture is observed and (iv) isolatingfrom the culture the bacteriophage. In still other embodiments, theinvention provides for a method of producing and/or isolating abacteriophage having a genome that comprises or consists of the nucleicacid sequence of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7comprising (i) obtaining a culture of S. aureus, (ii) infecting it withthe bacteriophage having a genome comprising or consisting of thenucleic acid sequence of ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ IDNO:7; (iii) culturing until significant lysis of the culture is observedand (iv) isolating from the culture the bacteriophage. In yet stillother embodiments, the invention provides for a method of producingand/or isolating a bacteriophage having a genome that comprises orconsists of the nucleic acid sequence of SEQ ID NO:760 comprising (i)obtaining a culture of A. baumannii, (ii) infecting it with thebacteriophage having a genome comprising or consisting of the nucleicacid sequence of SEQ ID NO:760; (iii) culturing until significant lysisof the culture is observed and (iv) isolating from the culture thebacteriophage.

Bacteriophage may be isolated from a bacterial sample using any methoddescribed herein or known in the art (see, e.g., Carlson, “Working withbacteriophages: common techniques and methodological approaches,” In,Kutter and Sulakvelidze (Eds) Bacteriophages: Biology and Applications,5^(th) ed. CRC Press (2005); incorporated herein by reference in itsentirety).

The invention also provides for polypeptides isolated frombacteriophages of the invention. The isolated polypeptides may be fulllength bacteriophage proteins or may be fragments, variants orderivatives of the bacteriophage proteins provided that the fragment,variant or derivative exhibit at least one biological activityassociated with the bacteriophage or polypeptide from which it isderived. In certain embodiments, the polypeptides of the invention areisolated from bacteriophage F1245/05 (which typically infects A.baumannii), F168/08 or F170/08 (which typically infects E. faecalisand/or E. faecium), bacteriophage F770/05 (which typically infects P.aeruginosa) or bacteriophage F197/08, F86/06, F87s/06 or F91a/06 (whichtypically infect S. aureus).

In specific embodiments, the polypeptide of the invention is anendolysin or lysin isolated from a bacteriophage having a genomecomprising or consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:760 (e.g., bacteriophageF168/08, F170/08, F197/08, F86/06, F87s/06, F91a/06, or F1245/05respectively). In specific embodiments, the polypeptide of the inventionis an endolysin or lysin having the amino acid sequence comprising orconsisting of SEQ ID NO:797, SEQ ID NO:68, SEQ ID NO:184, SEQ ID NO:202,SEQ ID NO:203, SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQ IDNO:575, SEQ ID NO:641 or SEQ ID NO:712. In other embodiments, theisolated polypeptide of the invention is a fragment, variant orderivative of an endolysin or lysin isolated from a bacteriophage of theinvention, which fragment, variant or derivative exhibits at least onebiological activity, preferably antibacterial activity (e.g., lytickilling activity), of the endolysin, lysin or bacteriophage from whichit is isolated or derived. Accordingly, in certain embodiments, theinvention provides isolated polypeptides that are fragments, variants orderivatives of endolysins or lysins isolated from bacteriophages of theinvention, which fragments, variants or derivatives exhibitantibacterial or antimicrobial activity (e.g., lytic killing activity)against one or more of A. baumannii, E. faecalis, E. faecium or S.aureus. In other embodiments, the isolated polypeptides that arefragments, variants or derivatives of endolysins or lysins isolated frombacteriophages of the invention that exhibit antibacterial orantimicrobial activity (e.g., lytic killing activity) against one ormore species of bacteria other than A. baumannii, E. faecalis, E.faecium or S. aureus (e.g., P. aeruginosa). In certain embodiments, thepolypeptide of the invention comprises or consists of the amino acidsequence SEQ ID NO:68, SEQ ID NO:184, SEQ ID NO:202 or SEQ ID NO:203, ora fragment, variant or derivative thereof, which polypeptide exhibitsantibacterial or antimicrobial activity against E. faecalis or E.faecium. In other embodiments, the polypeptide of the inventioncomprises or consists of the amino acid sequence SEQ ID NO:446, SEQ IDNO:447, SEQ ID NO:448, SEQ ID NO:575, SEQ ID NO:641 or SEQ ID NO:712, ora fragment, variant or derivative thereof, which polypeptide exhibitsantibacterial or antimicrobial activity against S. aureus. In yet stillother embodiments, the polypeptide of the invention comprises orconsists of the amino acid sequence SEQ ID NO:797, or a fragment,variant or derivative thereof, which polypeptide exhibits antibacterialor antimicrobial activity against A. baumanni.

In certain embodiments, the polypeptide of the invention comprises orconsists of a CHAP domain isolated from an endolysin or lysin ofbacteriophage F168/08, F170/08, F770/05, F197/08, F86/06, F87s/06 orF91a/06. Isolated CHAP domains have been demonstrated to retain theantibacterial activity, e.g., lytic killing activity, of the endolysinor lysin from which they are derived; CHAP domains may be identified andisolated by methods routine in the art (see, e.g., Rigden et al., 2003,Trends Biochem. Sci. 28:230-234; Bateman et al., 2003, Trends Biochem.Sci. 28:234-237, each of which is incorporated by reference herein inits entirety). In specific embodiments, the polypeptide of the inventioncomprises or consists of a CHAP domain isolated from a polypeptidehaving an amino acid sequence of SEQ ID NO:68, SEQ ID NO:184, SEQ IDNO:202, SEQ ID NO:203, SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQID NO:575, SEQ ID NO:641 or SEQ ID NO:712. In specific embodiments, theinvention provides for an isolated polypeptide that comprises orconsists of the CHAP domain derived from a second polypeptide having theamino acid sequence of SEQ ID NO:68, SEQ ID NO:446, SEQ ID NO:575, SEQID NO:641 or SEQ ID NO:712, wherein the CHAP domain has the amino acidsequence of SEQ ID NO:755, SEQ ID NO:756, SEQ ID NO:757, SEQ ID NO:758or SEQ ID NO:759, respectively. That is, SEQ ID NO:755 corresponds to aCHAP domain derived from the polypeptide having amino acid sequence ofSEQ ID NO:68; SEQ ID NO:756 corresponds to a CHAP domain derived fromthe polypeptide having amino acid sequence of SEQ ID NO:446, and soforth. In other embodiments the invention provides for a fragment,variant or derivative of a CHAP domain of isolated from an endolysin orlysin of bacteriophage F168/08, F170/08, F770/05, F197/08, F86/06,F87s/06 or F91a/06, which fragment, variant or derivative exhibits atleast one biological activity, e.g., lytic cell killing, of the CHAPdomain from which it was derived and wherein said CHAP domain has anamino acid sequence of SEQ ID NO:755, SEQ ID NO:756, SEQ ID NO:757, SEQID NO:758 or SEQ ID NO:759. The amino acid sequences of SEQ IDNO:755-SEQ ID NO:759 are provided in Table 1.

TABLE 1 Amino acid sequences of CHAP domains isolated frombacteriophages of the invention SEQ ID NO Sequence Phage 755NGLVGKGVDADGWYGTQCMDLTVDVMQRFFGWRPYGNAIALV F168/08DQPIPAGFQRIRTTSSTQIKAGDVMIWGLGYYAQYGHTGIATEDGRADGTFVSVDQNWINPSLEVGSPAAAIHHNMDGVWGVIR 756DNSLGKQFNPDLFYGFQCYDYANMFFMIATGERLQGLYAYNIPF F197/08DNKARIEKYGQIIKNYDSFLPQKLDIVVFPSKYGGGAGHVEIVESANLNTFTSYGQNWNGKGWTNGVAQPGWGPETVTRHVHYYDDPM YFIR 757RWYQGRYIDFDGWYGYQCADLAVDYIYWLLEIRMWGNAKDAI F86/06NNDFKNMATVYENTPSFVPQIGDVAVFTKGIYKQYGHIGLVFNGGNTNQFLILEQNYDGNANTPAKLRWDNYYGCTHFIR 758RWYQGRYIDFDGWYGYQCADLAVDYIYWLLEIRMWGNAKDAI F87s/06NNDFKNMATVYENTPSFVPQIGDVAVFTKGIYKQYGHIGLVFNGGNTNQFLILEQNYDGNANTPAKLRWDNYYGCTHFIR 759RWYQGRYIDFDGWYGYQCADLAVDYIYWLLEIRMWGNAKDAI F91a/06NNDFKNMATVYENTPSFVPQIGDVAVFTKGIYKQYGHIGLVFNGGNTNQFLILEQNYDGNANTPAKLRWDNYYGCTHFIR

In certain embodiments, a polypeptide of the invention comprises orconsists of a tail length tape measure protein or tail protein (e.g.,tail component, tail fiber protein, adsorption associated tail protein),or fragment thereof, isolated from a bacteriophage having a genomecomprising or consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:760 (e.g.,bacteriophage F168/08, F170/08, F770/05, F197/08, F86/06, F87s/06,F91a/06, or F1245/05, respectively), wherein the tail length tapemeasure protein or tail protein, or fragment thereof has a biologicfunction associated with the bacteriophage from which it is derived,e.g., antimicrobial or antibacterial activity (e.g., lytic killingactivity). In specific embodiments, the antimicrobial or antibacterialactivity of the tail length tape measure protein or tail protein isdirected against at least one or more species or strains of A.baumannii, E. faecalis, E. faecium, P. aeruginosa and/or S. aureus. Inspecific embodiments, the polypeptide of the invention is a tail tapemeasure protein or tail protein having the amino acid sequencecomprising or consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:204,SEQ ID NO:214, SEQ ID NO:435, SEQ ID NO:438, SEQ ID NO:440, SEQ IDNO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534,SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ IDNO:539, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:632, SEQ ID NO:633, SEQID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704,or SEQ ID NO:795. In other embodiments, the isolated polypeptide of theinvention is a fragment, variant or derivative of the amino acidsequence of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:204, SEQ ID NO:214,SEQ ID NO:435, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:525, SEQ IDNO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535,SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ IDNO:567, SEQ ID NO:568, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:700, SEQID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, or SEQ IDNO:795, which fragment variant or derivative exhibits at least onebiological activity or function of the bacteriophage from which it isisolated or derived, e.g., antimicrobial or antibacterial activity(e.g., lytic killing activity). In preferred embodiments, the at leastone biological activity or function of the fragment, variant orderivative is directed against one or more strains of E. faecalis, E.faecium, P. aeruginosa, S. aureus, and/or A. baumannii.

In certain embodiments, the isolated polypeptide of the invention is avariant of a bacteriophage polypeptide, which variant comprises orconsists of a amino acid sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or greater sequence identity to a second amino acid sequenceof the same length (i.e., consisting of the same number of residues),which second amino acid sequence is SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:68, SEQ ID NO:184, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQID NO:214, SEQ ID NO:435, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:446,SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:525, SEQ ID NO:526, SEQ IDNO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536,SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:567, SEQ IDNO:568, SEQ ID NO:575, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:641, SEQID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704,SEQ ID NO:712, SEQ ID NO:755, SEQ ID NO:756, SEQ ID NO:757, SEQ IDNO:758, SEQ ID NO:759, SEQ ID NO:795, or SEQ ID NO:797, and/or afragment thereof, and wherein the variant exhibits at least onebiological function or activity of the bacteriophage from which it wasderived (e.g., antimicrobial or antibacterial activity (e.g., lytickilling activity)) against one or more strains of bacteria (e.g.,Gram-positive bacteria (e.g., E. faecalis, E. faecium, S. aureus),Gram-negative bacteria (e.g., P. aeruginosa, A baumannii) or bacterianot classified as either Gram-positive or Gram-negative).

In certain embodiments, the invention provides an isolated polypeptidehaving an amino acid sequence of any of SEQ ID NOS:2-124, SEQ ID NOS:126-338, SEQ ID NOS:340-434, SEQ ID NOS:436-550, SEQ ID NOS:552-614, SEQID NOS:616-680, SEQ ID NOS:682-759, SEQ ID NOS:761-816, and activebiologic fragments thereof. In preferred embodiments, the variantpolypeptide of the invention exhibits at least one biologic activityassociated with the polypeptide or bacteriophage from which it wasisolated or derived directed against at least one or more strains of E.faecalis, E. faecium, P. aeruginosa, S. aureus, and/or A. baumannii.

In other embodiments, the invention provides an isolated nucleic acidsequence encoding the amino acid sequence of one of SEQ ID NOS:8-130,SEQ ID NOS:131-343, SEQ ID NOS:344-438, SEQ ID NOS:439-553, SEQ IDNOS:554-616, SEQ ID NOS:617-681, SEQ ID NOS:682-759, SEQ ID NOS:761-816,and active fragments thereof. In other embodiments the inventionprovides the nucleic acid sequence of any of the open reading framesidentified in FIGS. 2, 5, 8, 11, 14, 17, 20, and 23.

In certain embodiments, the polypeptides of the present invention arerecombinantly fused or chemically conjugated (including both covalentlyand non-covalently conjugations) to therapeutic agents, e.g.,heterologous polypeptides or small molecules, to generate fusionproteins or chimeric polypeptides. The fusion does not necessarily needto be direct, but may occur through linker sequences or through chemicalconjugation. Non-limiting examples of therapeutic agents to which thepolypeptides of the invention may be conjugated are peptide ornon-peptide cytotoxins (including antimicrobials and/or antibiotics),tracer/marker molecules (e.g., radionuclides and fluorphores) and otherantibiotic or antibacterial compounds known in the art.

6.1 Antibiotic Compositions

The isolated bacteriophages or polypeptides of the present invention maybe administered alone or incorporated into a pharmaceutical compositionfor the use in treatment or prophylaxis of bacterial infections, e.g.,infections caused by bacteria including, but not limited to, A.baumannii, E. faecalis, E, faecium, P. aeruginosa and S. aureus. Thepolypeptides may be combined with a pharmaceutically acceptable carrier,excipient, or stabilizer. Examples of pharmaceutically acceptablecarriers, excipients and stabilizers include, but are not limited to,buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin and gelatin; hydrophilic polymers suchas polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Thepharmaceutical composition of the present invention (e.g., antibacterialcomposition) can also include a lubricant, a wetting agent, a sweetener,a flavoring agent, an emulsifier, a suspending agent, and apreservative, in addition to the above ingredients.

The bacteriophages and/or polypeptides of the present invention may alsobe combined with one or more therapeutic and or prophylactic agentsuseful for the treatment of bacterial infection as described hereinand/or known in the art (e.g. one or more lysins). The pharmaceuticalcompositions of the invention may therefore comprise two or moreisolated bacteriophages of the invention (with antibacterial activityagainst the same or different bacterial species or strains), thecombination of a bacteriophage and a polypeptide of the invention or thecombination of a bacteriophage and/or polypeptide of the invention and abacteriophage and/or therapeutic polypeptide known in the art. Inspecific embodiments, the therapeutic components of a combination targettwo or more species or strains of bacteria or exhibit differingenzymatic activity. For example, lysins, in general, exhibit one ofamidase, endopeptidase, muramidase or glucosamidase activity.Accordingly, the combination of lysins exhibiting different activitiesmay provide synergistic enhancement to the therapeutic activity of thepharmaceutical composition of the invention.

Examples of other therapeutic agents that may be used in combinationwith the polypeptide of the invention include, but are not limited to,standard antibiotic agents, anti-inflammatory agents, antiviral agents,local anesthetic agents, and corticosteroids.

Standard antibiotics that maybe used with pharmaceutical compositionscomprising polypeptides of the invention include, but are not limitedto, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin,rhodostreptomycin, streptomycin, tobramycin, apramycin, rifamycin,naphthomycin, mupirocin, geldanamycin, ansamitocin, carbacephems,imipenem, meropenem, ertapenem, faropenem, doripenem,panipenem/betamipron, biapenem, PZ-601, cephalosporins, cefacetrile,cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine,cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin,cefradine, cefroxadine, ceftezole, cefaclor, cefonicid, cefprozil,cefuroxime, cefuzonam, cefinetazole, cefotetan, cefoxitin, cefcapene,cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime,cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone,cefoperazone, ceftazidime latamoxef, cefclidine, cefepime, cefluprenam,cefoselis, cefozopran, cefpirome, cefquinome, flomoxef. ceftobiprole,azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, aztreonam, pencillin and penicillin derivatives,actinomycin, bacitracin, colistin, polymyxin B, cinoxacin, flumequine,nalidixic acid, oxolinic acid, piromidic acide, pipemidic acid,rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin,nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin,balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin,pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin,garenoxacin, gemifloxacin, stifloxacin, trovalfloxacin, prulifloxacin,acetazolamide, benzolamide, bumetanide, celecoxib, chlorthalidone,clopamide, dichlorphenamide, dorzolamide, ethoxyzolamide, furosemide,hydrochlorothiazide, indapamide, mafendide, mefruside, metolazone,probenecid, sulfacetamide, sulfadimethoxine, sulfadoxine,sulfanilamides, sulfamethoxazole, sulfasalazine, sultiame, sumatriptan,xipamide, tetracycline, chlortetracycline, oxytetracycline, doXycycline,lymecycline, meclocycline, methacycline, minocycline, rolitetracyclineand any combination thereof in amounts that are effective to additivelyor synergistically enhance the therapeutic effect of the bacteriophageor polypeptide of the invention for a given infection.

Local anesthetics that may be used in pharmaceutical compositions of thepresent invention include tetracaine, tetracaine hydrochloride,lidocain, lidocaine hydrochloride, dimethisoquin hydrochloride,dibucaine, dibucaine hydrochloride, butambenpicrate, and pramoxinehydrochloride. An exemplary concentration of local anesthetic is about0.025% to about 5% by weight of the total composition.

Corticosteroids that may be useful in combination with the polypeptides,phage, and/or pharmaceutical compositions of the invention includebetamethasone, dipropionate, fluocinolone, actinide, betamethasonevalerate, triamcinolone actinide, clobetasol propionate, desoximetasone,diflorasone diacetate, amcinonide, flurandrenolide, hydrocortisonevalerate, hydrocortisone butyrate, and desonide. An exemplaryconcentration of corticosteroid is about 0.01% to about 1% by weight ofthe total composition.

In certain embodiments, a formulation comprising a bacteriophage and/orpolypeptide of the invention further comprises SM buffer (0.05 MTris-HCl (pH 7.4-7.5); 0.1 M NaCl; 10 mM MgSO₄). In other embodiments,the formulation further comprises SM buffer and 10 mM MgCl₂. In stillother embodiments, the formulation further comprises SM buffer and about20% or about 30% ethanol.

Pharmaceutical compositions comprising a bacteriophage and/orpolypeptide of the present invention can be formulated in a unit dose ormulti-dose formulation. Suitable formulations can be selected from thegroup consisting of ointments, solutions, suspensions or emulsions,extracts, powders, granules, sprays, lozenges, tablets or capsules andadditionally include a dispersing agent or a stabilizing agent.

The pharmaceutical compositions of the invention can be administered byinhalation, in the form of a suppository or pessary, topically (e.g., inthe form of a lotion, solution, cream, ointment or dusting powder), epi-or transdermally (e.g., by use of a skin patch), orally (e.g., as atablet, which may contain excipients such as starch or lactose), as acapsule, ovule, elixirs, solutions or suspensions (each optionallycontaining flavoring, coloring agents and/or excipients), or they can beinjected parenterally (e.g., intravenously, intramuscularly orsubcutaneously). For parenteral administration, the compositions may bebest used in the form of a sterile aqueous solution which may containother substances, for example enough salts or monosaccharides to makethe solution isotonic with blood. For buccal or sublingualadministration the compositions may be administered in the form oftablets or lozenges which can be formulated in a conventional manner. Ina preferred embodiment, a bacteriophage and/or polypeptide of thepresent invention is administered topically, either as a single agent,or in combination with other antibiotic treatments as described hereinor known in the art.

A bacteriophage and/or polypeptide of the present invention may also bedermally or transdermally administered. For topical application to theskin, the bacteriophages and/or polypeptides of the present inventionmay be combined with one, or a combination of carriers, which includebut are not limited to, an aqueous liquid, an alcohol base liquid, awater soluble gel, a lotion, an ointment, a nonaqueous liquid base, amineral oil base, a blend of mineral oil and petrolatum, lanolin,liposomes, proteins carriers such as serum albumin or gelatin, powderedcellulose carmel, and combination thereof. A topical mode of deliverymay include a smear, a spray, a time-release patch, a liquid absorbedwipe, and combinations thereof. The bacteriophage and/or polypeptide ofthe invention may be applied to a patch or bandage either directly or inone of the carriers. The patches may be damp or dry, wherein the phageand/or polypeptide (e.g., a lysin) is in a lyophilized form on thepatch. The carriers of topical compositions may comprise semi-solid andgel-like vehicles that include a polymer thickener, water,preservatives, active surfactants, or emulsifiers, antioxidants, sunscreens, and a solvent or mixed solvent system. U.S. Pat. No. 5,863,560discloses a number of different carrier combinations that can aid in theexposure of skin to a medicament, and its contents are incorporatedherein.

For intranasal or administration by inhalation, the bacteriophage and/orpolypeptide of the invention is conveniently delivered in the form of adry powder inhaler or an aerosol spray presentation from a pressurizedcontainer, pump, spray or nebuliser with the use of a suitablepropellant, e.g. dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A.™.) or1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA.™.), carbon dioxide or othersuitable gas. In the case of a pressurized aerosol, the dosage unit maybe determined by providing a valve to deliver a metered amount. Thepressurized container, pump, spray or nebuliser may contain a solutionor suspension of the active compound, e.g. using a mixture of ethanoland the propellant as the solvent, which may additionally contain alubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, forexample, from gelatin) for use in an inhaler or insufflator may beformulated to contain a powder mix of the bacteriophage and/orpolypeptide of the invention and a suitable powder base such as lactoseor starch.

For administration in the form of a suppository or pessary, thetherapeutic compositions may be applied topically in the form of a gel,hydrogel, lotion, solution, cream, ointment or dusting powder.Compositions of the invention may also be administered by the ocularroute. For ophthalmic use, the compositions of the invention can beformulated as micronized suspensions in isotonic, pH adjusted, sterilesaline, or, preferably, as solutions in isotonic, pH adjusted, sterilesaline, optionally in combination with a preservative such as abenzylalkonium chloride. Alternatively, they may be formulated in anointment such as petrolatum.

Dosages and desired drug concentrations of the pharmaceuticalcompositions of the present invention may vary depending on theparticular use. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments can provide reliable guidance for the determination ofeffective doses in human therapy. Interspecies scaling of effectivedoses can be performed by one of ordinary skill in the art following theprinciples described by Mordenti, J. and Chappell, W. “The use ofinterspecies scaling in toxicokinetics” in Toxicokinetics and New DrugDevelopment, Yacobi et al., Eds., Pergamon Press, New York 1989, pp42-96.

6.2 Therapeutic Use

The bacteriophages and polypeptides of the present invention haveactivity against a plurality of strains of E. faecalis, E. faecium, P.aeruginosa, S. aureus, and/or A. baumannii-, as described in FIGS. 3A-B,6A-B, 9, 12, 15, 18, 21, and 23. Therefore, the compositions of thepresent invention may used in methods of preventing and treatinginfections associated with E. faecalis, E. faecium, P. aeruginosa, S.aureus, and/or A. baumannii in both humans and animals. In otherembodiments, the compositions of the present invention may be used totreat infection associated with related species or strains of thesebacteria, including, but not limited to S. epidermidis, S. auricularis,S. capitis, S. haemolyticus, S. hominis, S. saprophyticus, S. simulans,S. xylosis, Micrococcus luteus, Bacilus subtilis, B. pumilus, E. hirae,and/or one or more of the strains of A. baumannii, e.g, one or more ofthe strains described in FIG. 24.

In specific embodiments, the subject receiving a pharmaceuticalcomposition of the invention is a mammal (e.g., bovine, ovine, caprine,equid, primate (e.g., human), rodent, lagomorph or avian (e.g., chicken,duck, goose)). In the context of the present invention, “treatment”refers to therapeutic treatment and wherein the object is to eliminate,lessen, decrease the severity of, ameliorate, slow the progression of orprevent the symptoms or underlying cause (e.g., bacterial infection)associated with the pathological condition or disorder. “Treatment”refers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to eliminate, lessen, decrease theseverity of, slow the progression of or delay or prevent the symptoms orunderlying cause (e.g., bacterial infection) associated with thepathological condition or disorder. It is also contemplated that abacteriophage and/or polypeptide of the invention acts as a prophylacticor preventative measure, preventing the onset of infection caused by oneor more bacteria.

A. baumannii, E. faecalis, E. faecium, P. aeruginosa and S. aureus areresponsible for many severe opportunistic infections, particularly inindividuals with compromised immune systems. The pharmaceuticalcompositions of the present invention are contemplated for treating anyinfection associated with A. baumannii, E. faecalis, E. faecium, P.aeruginosa or S. aureus, or associated with other species or strains ofbacteria, including, but not limited to, infections of the skin(including but not limited to skin ulcers, bed sores and diabetic footulcers), infections in and around wounds, post-operative infections,infections associated with catheters and surgical drains and infectionsof the blood.

A. baumannii, E. faecalis, E. faecium, P. aeruginosa and S. aureus arealso associated with infections that involve organ systems that have ahigh fluid content, and it is contemplated that the bacteriophagesand/or polypeptides of the invention have therapeutic use in theprevention and treatment of these infections. For example, thepharmaceutical compositions of the invention may be used for theprevention or treatment of infections of the respiratory tract, of thecerebrospinal fluid, of peritoneal fluid, and of the urinary tract. Thecompositions of the invention may also be used to prevent and/or treatnosocomial pneumonia, infections associated with continuous ambulatoryperitoneal dialysis (CAPD), catheter-associated bacteruria, andnosocomial meningitis.

In a preferred embodiment, a bacteriophage and/or polypeptide of theinvention is used prophylactically in hospital setting, particularly toprevent infections associated with wounds, ulcers, and openings in theskin due to catheterization, and any other medical procedures ordevices.

In certain embodiments, a bacteriophage and/or polypeptide of theinvention is used as a single agent for treating or preventinginfections associated with A. baumannii, E. faecalis, E. faecium, P.aeruginosa, S. aureus or other bacterial species. In other embodimentsof the invention, a bacteriophage and/or polypeptide of the invention isused in combination with other agents, including other bacteriophages(for example, that target a different species or strain of bacteria), orwith antibiotics that target the same or different kinds of bacteria,including bacteria selected from any gram-positive bacteria, anygram-negative bacteria, and any other groups of bacteria that is notclassified as gram-positive or gram-negative. The compositions of theinvention may also be used in combination with any other means oftreating bacterial infection known to one of skill in the art.

Also contemplated by the invention are methods of preventing and methodsof treating an infection caused by bacteria including, but not limitedto, E. faecalis, E. faecium, P. aeruginosa, S. aureus, and/or A.baumannii, comprising administering to a mammal in need thereof acomposition comprising a lysin comprising or consisting of the aminoacid sequence of SEQ ID NO:68, SEQ ID NO:184, SEQ ID NO:202, SEQ IDNO:203, SEQ ID NO:446, SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:575, SEQID NO:641, I.D. NO:712, and/or SEQ ID NO:797, or a fragment, variant orderivative thereof, wherein the fragment, variant or derivative exhibitsantibacterial or antimicrobial activity against the species of bacteriafrom which the parent bacteriophage was isolated. In a specific examplein accordance with this embodiment, the invention provides methods ofpreventing or treating an infection caused by a bacteria including, butnot limited to, E. faecalis, E. faecium, P. aeruginosa, S. aureus,and/or A. baumannii, comprising administering to a mammal in needthereof a composition comprising an isolated CHAP domain of a lysin, ora fragment, variant or derivative thereof that exhibits at least onebiologic activity of the CHAP domain from which it was isolated (e.g.,lytic cell killing). In certain embodiments, the isolated CHAP domaincomprises or consists of the amino acid sequence of SEQ ID NO:755, SEQID NO:756, SEQ ID NO:757, SEQ ID NO:758 or SEQ ID NO:759. In otherembodiments, the invention provides methods of preventing and treatingan infection caused by bacteria including, but not limited to, E.faecalis, E. faecium, P. aeruginosa, S. aureus, and/or A. baumannii,comprising administering to a mammal in need thereof a compositioncomprising a tail tape measure protein or tail protein comprising orconsisting of the amino acid sequence of SEQ ID NO:61, SEQ ID NO:63, SEQID NO:204, SEQ ID NO:214, SEQ ID NO:435, SEQ ID NO:438, SEQ ID NO:440,SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ IDNO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538,SEQ ID NO:539, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:632, SEQ IDNO:633, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQID NO:704, and/or SEQ ID NO:795, or a fragment, variant or derivativethereof, wherein the fragment, variant or derivative exhibits a biologicactivity associated with the parent bacteriophage. In still otherembodiments, the invention provides methods of preventing and treatingan infection caused by bacteria including, but not limited to, E.faecalis, E. faecium, P. aeruginosa, S. aureus, and/or A. baumannii,comprising administering to a mammal in need thereof a compositioncomprising bacteriophage having a genome comprising or consisting of thenucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and/or SEQ ID NO:760.Combinations of the lysins (or fragments, variants or derivativesthereof as described above) and of tail tape measure proteins or tailproteins (or fragments, variants or derivatives thereof as describedabove), optionally with one or more bacteriophages of the invention orwith other treatments, such as antibiotics, are also contemplated, aswell as methods of treating and methods of preventing a bacterialinfection using one or more of the combinations herein described.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agent. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disease ordisorder. A first prophylactic or therapeutic agent can be administeredprior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes,15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second prophylactic or therapeutic agent (differentfrom the first prophylactic or therapeutic agent) to a subject with adisease or disorder.

6.3 Disinfectant and Anti-Infective Use

Bacterial pathogens most often infect at a mucous membrane site (e.g.,upper and lower respiratory, intestinal, urogenital and ocular). Themucous membranes themselves are often the reservoir, sometimes the onlyreservoir, for many pathogenic bacteria found in the environment (e.g.pneumococci, staphylococci and streptococci). There are very fewanti-infectives that are designed to control the carrier state ofpathogenic bacteria. However, studies have shown that by reducing oreliminating this reservoir in environments such as hospitals and nursinghomes, the incidence of infections by these bacteria will be markedlyreduced.

The bacteriophages and/or polypeptides of the present invention may beused in anti-infective compositions for controlling the growth ofbacteria (e.g., Gram-positive bacteria (e.g., E. faecalis, E. faecium,S. aureus), Gram-negative bacteria (e.g., P. aeruginosa, A. baumannii)or bacteria not classified as either Gram-positive or Gram-negative), inorder to prevent or reduce the incidence of serious infections. Inaddition to use in compositions for application to mucous membranes, abacteriophage and/or polypeptide of the present incorporation may alsobe incorporated into formulations such as gels, creams, ointments, orsprays for controlling or preventing colonization of bacteria on bodysurfaces (e.g., skin and mucus membranes) (e.g., for sterilization ofsurgical fields or of the hands and exposed skin of healthcare workersand/or patients) and other solid surfaces (e.g., appliances, countertopsand, in particular, hospital equipment).

6.4 Diagnostic Methods

The present invention also encompasses diagnostic methods fordetermining the causative agent in a bacterial infection. In certainembodiments, the diagnosis of the causative agent in a presentation ofbacterial infection is performed by (i) culturing tissue, blood or fluidsamples of a patient according to standard techniques, (ii) contactingthe culture with one or more bacteriophages and/or polypeptides of theinvention and (iii) monitoring cell growth and evidence of lysis aftersaid contacting. Because the activity of bacteriophages and/or theirisolated products (e.g., polypeptides, or biologically active fragments,variants or derivatives thereof) tends to be species or strain specific,susceptibility, or lack of susceptibility, to one or more bacteriophagesand/or polypeptides of the invention may be indicative of the species orstrain of infective bacteria. For example, decreased growth of testcultures after contacting with a bacteriophage having a genomecomprising or consisting of the nucleic acid sequence SEQ ID NO:1 or SEQID NO:2, or with an isolated polypeptide product thereof, may beindicative of the test sample comprising E. faecalis or E. faecium.Similarly, a bacteriophage having a genome comprising or consisting ofthe nucleic acid sequence SEQ ID NO:3, or an isolated polypeptideproduct thereof, may be used to identify infection by P. aeruginosa; abacteriophage having a genome comprising or consisting of the nucleicacid sequence SEQ ID NO:760, or an isolated polypeptide product thereof,may be used to identify infection by A. baumannii, while that having agenome comprising or consisting of the nucleic acid sequence SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, or an isolatedpolypeptide product thereof, may be used to identify infection by S.aureus.

6.5 Amino Acid Variants

Amino acid sequence variants of the polypeptides of the invention can becreated such that they are substitutional, insertional or deletionvariants. Deletion variants lack one or more residues of the nativeprotein which are not essential for function (e.g., antimicrobial orantibacterial activity). Insertional mutants typically involve theaddition of material at a non-terminal point in the polypeptide.Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Substitutions of this kind preferably are conservative, thatis, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

Once general areas of the gene are identified as encoding the particularlysin protein as described herein, point mutagenesis may be employed toidentify with particularity which amino acid residues are important inthe antibacterial activities. Thus, one of skill in the art will be ableto generate single base changes in the DNA strand to result in analtered codon and a missense mutation.

Preferably, mutation of the amino acids of a protein creates anequivalent, or even an improved, second-generation molecule. Forexample, certain amino acids may be substituted for other amino acids ina protein structure without detectable loss of function (e.g.,antibacterial or antimicrobial activity). In making such changes, thehydropathic index of amino acids may be considered. The importance ofthe hydropathic amino acid index in conferring interactive biologicfunction on a protein is generally understood in the art. It is acceptedthat the relative hydropathic character of the amino acid contributes tothe secondary structure of the resultant protein, which in turn definesthe interaction of the protein with other molecules, for example,interaction with a peptidoglycan within the outer coat of agram-positive bacteria. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics;for example: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan 0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. Likehydrophobicity, values of hydrophilicity have been assigned to eachamino acid: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1);glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5+1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). Equivalent molecules may be obtained bysubstitution of one amino acid for another where their hydrophilicityindices are within ±2, preferably ±1, or most preferably ±5 of eachother. In certain embodiments, the invention encompasses isolatedpeptides that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more aminoacid modifications (e.g., insertion, substitution, deletion, etc.)relative to an amino acid sequence disclosed herein. In preferredembodiments, the mutation(s) are made such that biological activity ofthe particular polypeptide is retained. For example, the presentinvention encompasses polypeptides isolated from bacteriophage F1245/05,F168/08, F170/08, F770/05, F197/08, F86/06, F87s/06 and/or F91a/06,which are mutated to comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or moreamino acid modifications relative to an amino acid sequence listedherein, and which exhibit antibacterial activity against one or morespecies or strains of Gram-positive or Gram-negative bacterium, e.g., A.baumannii, E. faecalis, E. faecium, P. aeruginosa and/or S. aureus. Inspecific embodiments, the polypeptides of the invention derived fromF168/08 or F/170/08 exhibit antibacterial or antimicrobial activity,e.g., lytic killing activity, against at least E. faecalis and/or E.faecium; those derived from F770/05 against at least P. aeruginosa;those derived from F197/08, F86/06, F87s/06 or F91a/06 against at leastS. aureus; and those derived from F1245/05 against at least A.baumannii.

6.6 Polynucleotides Encoding Polypeptides of the Invention

The invention provides polynucleotides comprising a nucleotide sequenceencoding a polypeptide of the invention. The invention also encompassespolynucleotides that hybridize under high stringency, intermediate orlower stringency hybridization conditions, e.g., as defined supra, topolynucleotides that encode a polypeptide of the invention and thatencode modified polypeptides that have antibiotic and/or otherbiological activity.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,a polynucleotide encoding a polypeptide of the invention may begenerated from nucleic acid from a suitable source (e.g., bacteriophageF1245/05, F168/08, F170/08, F770/05, F197/08, F197/08, F86/06, F87s/06or F91a/06). Nucleotide sequences may be isolated from phage genomes byroutine methods known in the art (see, e.g., Carlson, “Working withbacteriophages: common techniques and methodological approaches,” In,Futter and Sulakvelidze (Eds) Bacteriophages: Biology and Applications,5^(th) ed. CRC Press (2005); incorporated herein by reference in itsentirety). If a source containing a nucleic acid encoding a particularpolypeptide is not available, but the amino acid sequence of thepolypeptide of the invention is known, a nucleic acid encoding thepolypeptide may be chemically synthesized and cloned into replicablecloning vectors using any method well known in the art.

Once the nucleotide sequence of the polypeptide of the invention isdetermined, the nucleotide sequence of the polypeptide may bemanipulated using methods well known in the art for the manipulation ofnucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate polypeptides having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In yet another embodiment of the invention, the following nucleotidesequences are provided: the nucleotide sequence from nucleotide 1 tonucleotide 85 of SEQ ID NO:760; the nucleotide sequence from nucleotide87 to nucleotide 584 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 594 to nucleotide 767 of SEQ ID NO:760; the nucleotidesequence from nucleotide 724 to nucleotide 1035 of SEQ ID NO:760; thenucleotide sequence from nucleotide 1005 to nucleotide 1823 of SEQ IDNO:760; the nucleotide sequence from nucleotide 1816 to nucleotide 2130of SEQ ID NO:760; the nucleotide sequence from nucleotide 2132 tonucleotide 2383 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 2383 to nucleotide 3690 of SEQ ID NO:760; the nucleotidesequence from nucleotide 3687 to 4469 of SEQ ID NO:760; the nucleotidesequence from nucleotide 4466 to nucleotide 5458 of SEQ ID NO:760; thenucleotide sequence from nucleotide 5632 to nucleotide 7956 of SEQ IDNO:760; the nucleotide sequence from nucleotide 8010 to nucleotide 8912of SEQ ID NO:760; the nucleotide sequence from nucleotide 8915 tonucleotide 9262 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 9252 to nucleotide 10223 of SEQ ID NO:760; the nucleotidesequence from nucleotide 10213 to nucleotide 10782 of SEQ ID NO:760; thenucleotide sequence from nucleotide 10769 to nucleotide 11218 of SEQ IDNO:760; the nucleotide sequence from nucleotide 11202 to nucleotide11420 of SEQ ID NO:760; the nucleotide sequence from nucleotide 11413 tonucleotide 12342 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 12339 to nucleotide 12515 of SEQ ID NO:760; the nucleotidesequence from nucleotide 12512 to nucleotide 13165 of SEQ ID NO:760; thenucleotide sequence from nucleotide 13170 to nucleotide 15599 of SEQ IDNO:760; the nucleotide sequence from nucleotide 15609 to nucleotide15872 of SEQ ID NO:760; the nucleotide sequence from nucleotide 15979 tonucleotide 16173 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 16175 to nucleotide 16482 of SEQ ID NO:760; the nucleotidesequence from nucleotide 16494 to nucleotide 18059 of SEQ ID NO:760; thenucleotide sequence from nucleotide 18072 to nucleotide 18815 of SEQ IDNO:760; the nucleotide sequence from nucleotide 18857 to nucleotide19879 of SEQ ID NO:760; the nucleotide sequence from nucleotide 19930 tonucleotide 20178 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 20180 to nucleotide 20545 of SEQ ID NO:760; the nucleotidesequence from nucleotide 20646 to nucleotide 21203 of SEQ ID NO:760; thenucleotide sequence from nucleotide 21212 to nucleotide 23506 of SEQ IDNO:760; the nucleotide sequence from nucleotide 23506 to nucleotide24186 of SEQ ID NO:760; the nucleotide sequence from nucleotide 24201 tonucleotide 27068 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 27084 to nucleotide 30212 of SEQ ID NO:760; the nucleotidesequence from nucleotide 30214 to nucleotide 32505 of SEQ ID NO:760; thenucleotide sequence from nucleotide 32515 to nucleotide 32880 of SEQ IDNO:760; the nucleotide sequence from nucleotide 32873 to nucleotide33460 of SEQ ID NO:760; the nucleotide sequence from nucleotide 33460 tonucleotide 33816 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 33825 to nucleotide 35777 of SEQ ID NO:760; the nucleotidesequence from nucleotide 35774 to nucleotide 35872 of SEQ ID NO:760; thenucleotide sequence from nucleotide 35869 to nucleotide 36027 of SEQ IDNO:760; the nucleotide sequence from nucleotide 36038 to nucleotide36193 of SEQ ID NO:760; the nucleotide sequence from nucleotide 36916 tonucleotide 36788 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 37209 to nucleotide 37427 of SEQ ID NO:760; the nucleotidesequence from nucleotide 37868 to nucleotide 38386 of SEQ ID NO:760; thenucleotide sequence from nucleotide 38383 to nucleotide 38586 of SEQ IDNO:760; the nucleotide sequence from nucleotide 38912 to nucleotide39406 of SEQ ID NO:760; the nucleotide sequence from nucleotide 39406 tonucleotide 39915 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 39917 to nucleotide 40021 of SEQ ID NO:760; the nucleotidesequence from nucleotide 40018 to nucleotide 40101 of SEQ ID NO:760; thenucleotide sequence from nucleotide 40101 to nucleotide 40670 of SEQ IDNO:760; the nucleotide sequence from nucleotide 40720 to nucleotide41838 of SEQ ID NO:760; the nucleotide sequence from nucleotide 41822 tonucleotide 42127 of SEQ ID NO:760; the nucleotide sequence fromnucleotide 42105 to nucleotide 42308 of SEQ ID NO:760; the nucleotidesequence from nucleotide 42382 to nucleotide 42912 of SEQ ID NO:760; andthe nucleotide sequence from nucleotide 42896 to nucleotide 43015 of SEQID NO:760.

6.7 Recombinant Expression of Molecules of the Invention

Once a nucleic acid sequence encoding a molecule of the invention (e.g.,a polypeptide) has been obtained, the vector for the production of themolecules may be produced by recombinant DNA technology using techniqueswell known in the art. Methods which are well known to those skilled inthe art can be used to construct expression vectors containing thecoding sequences for the molecules of the invention and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, for example, thetechniques described in Sambrook et al., 1990, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. and Ausubel et al. eds., 1998, Current Protocols inMolecular Biology, John Wiley & Sons, NY).

The present invention provides expression vectors encoding thepolypeptides of the invention. An expression vector comprising thenucleotide sequence of a molecule identified by the methods of theinvention can be transferred to a host cell by conventional techniques(e.g., electroporation, liposomal transfection, and calcium phosphateprecipitation) and the transfected cells are then cultured byconventional techniques to produce the molecules of the invention. Inpreferred embodiments, the host cell is other than the species of theparent bacteria from which the bacteriophage comprising the sequence wasderived. In specific embodiments, the expression of the molecules of theinvention is regulated by a constitutive, an inducible or a tissue,specific promoter. In specific embodiments the expression vector ispQE-30 (Qiagen) or pET-29(a) (Novagen).

The host cells used to express the molecules identified by the methodsof the invention may be either bacterial cells (non susceptible to thebacteriophage protein or fragment thereof of the invention) such asEscherichia coli. A variety of host-expression vector systems may beutilized to express the molecules identified by the methods of theinvention. Such host-expression systems represent vehicles by which thecoding sequences of the molecules of the invention may be produced andsubsequently purified, but also represent cells which may, whentransformed or transfected with the appropriate nucleotide codingsequences, express the molecules of the invention in situ. Theseinclude, but are not limited to, microorganisms such as bacteria thatare not susceptible to the bacteriophage protein or fragment thereof ofthe invention (e.g., E. coli and B. subtilis) transformed withrecombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing coding sequences for the molecules identified by themethods of the invention; yeast (e.g., Saccharomyces pichia) transformedwith recombinant yeast expression vectors containing sequences encodingthe molecules identified by the methods of the invention; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the sequences encoding the molecules identifiedby the methods of the invention; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus(CaMV) and tobacco mosaic virus (TMV) or transformed with recombinantplasmid expression vectors (e.g., Ti plasmid) containing sequencesencoding the molecules identified by the methods of the invention; ormammalian cell systems (e.g., COS, CHO, BHI, 293, 293T, 3T3 cells,lymphotic cells (see U.S. Pat. No. 5,807,715), Per C.6 cells (humanretinal cells developed by Crucell) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems not susceptible to the bacteriophage protein orfragment of the invention, a number of expression vectors may beadvantageously selected depending upon the use intended for the moleculebeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of apolypeptide, vectors which direct the expression of high levels offusion protein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the proteinsequence may be ligated individually into the vector in frame with thelac Z coding region so that a fusion protein is produced; pIN vectors(Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke &Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectorsmay also be used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption andbinding to a matrix glutathione-agarose beads followed by elution in thepresence of free gluta-thione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The polypeptide coding sequence may becloned individually into non-essential regions (e.g., the polyhedringene) of the virus and placed under control of an AcNPV promoter (e.g.,the polyhedrin promoter).

Once a molecule of the invention (i.e., polypeptides) has beenrecombinantly expressed, it may be purified by any method known in theart for purification of polypeptides, for example, by chromatography(e.g., ion exchange, affinity, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of polypeptides or antibodies.

Certain modifications and improvements will occur to those skilled inthe art upon a reading of the foregoing description. It should beunderstood that all such modifications and improvements have beendeleted herein for the sake of conciseness and readability but areproperly within the scope of the following claims.

7. EXAMPLES

It is understood that the following examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggestive to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. All publications,patents, and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

Unless otherwise indicated, the bacteriophages of the invention wereisolated, processed and analyzed according to the following methods.

7.1 Methods

7.1.1 Purification of Phage.

Stock preparations of bacteriophage isolated from clinical samples wereprepared according to protocols described in Carlson, “Working withbacteriophages: common techniques and methodological approaches,” In,Kutter and Sulakvelidze (Eds) Bacteriophages: Biology and Applications,5^(th) ed. CRC Press (2005) (“Carlson,” incorporated herein by referencein its entirety).

The bacteriophage stock preparations were concentrated by precipitationwith PEG according to the protocol described Carlson and Yamamoto etal., 2004, PNAS 101:6415-6420. Briefly, the stock preparation wasincubated in 1 M NaCl for one hour at 4° C. with agitation. Next, PEG8000 (AppliChem, Cheshire, Mass.) was gradually added to a finalconcentration of 10% (w/v). The composition was then incubated overnightat 4° C. After the incubation period, the composition was centrifuged at10000×g for 30 minutes at 4° C. The sediment was then re-suspended in SMbuffer (0.05 M Tris-HCL at pH 7.4, 0.1 M NaCl, 10 mM MgSO₄) with gelatinat 1% w/v and centrifuged again at 1000 rpm at 4° C. for 10 minutes. Thesupernatant containing the suspended phage was saved for furtherpurification. The supernatant was purified using a CsCl gradientaccording to the methods in Carlson.

CsCl was removed from the purified and concentrated phage stock bydialysis. A dialysis membrane, Cellu.Sep H1 High Grade RegeneratedCellulose Tubular Membrane (Cellu.Sep, River Street, USA), was preparedaccording to the manufacturers' instructions. The dialysis consisted ofa first incubation of 30 minutes in 100 mM Tris-HCl and 3 M NaCl (pH7.4) at 4° C. This was followed by a second incubation of 30 minutes in100 mM Tris-HCl and 0.3 M NaCl (pH 7.4) at 4° C. After dialysis, thesuspended phage was removed from the interior of the dialysis bag andstored at 4° C.

7.1.2 Extraction of Phage Dna

To 5 ml of the purified and concentrated bacteriophage samples was added20 mM EDTA at pH 8.0, SDS at 0.5% (p/v) and Proteinase K at a finalconcentration of 40 μg/ml. The mixture was incubated at 56° C. for onehour. Successive extractions in phenol:chloroform:alcohol at aproportions of 25:24:1, were performed until the interface between theaqueous and organic phases was clear. The aqueous phase was then treatedwith an equal volume of chloroform and centrifuged at 13,0000×g for 10minutes at 4° C. The aqueous phase was once again removed, and the DNAwas precipitated by adding two volumes of absolute ethanol andincubating for thirty minutes at 20° C. The samples were thencentrifuged at 11,000×g for 30 minutes at 4° C. The pellet was washedwith 70% ethanol at room temperature and resuspended in 50 μl ofultra-pure water (Gibco, California). DNA concentration was determinedby measuring the absorbance at 260 nm in a ND-1000 Spectrophotometer.Integrity of the isolated phage DNA was analyzed by electrophoresis on a1% agarose gel.

7.1.3 Analysis of Bacteriophage Genomes

Sequencing of the bacteriophage genome allowed identification ofpotential open reading frames (ORFs) within the genome. The putativeORFs of the bacteriophages were used to search the NCBI nucleotidecollection database for homologous DNA sequences using the BLASTNprogram (see, e.g., Zhang et al., 2000, J. Comput. Biol. 7:203-214).

7.2 Example 1 Bacteriophage F168/08

Comparison of the putative ORFs of the bacteriophage F168/08 genome withthe sequences in the NCBI nucleotide database revealed that only smallportions of the genome (≦1%) exhibited homology with known sequences.The F168/08 ORFs, their encoded amino acid sequences and knownhomologous proteins are provided in FIG. 2. Prediction of orfs wasperformed by integrating the results obtained with GeneMark.hmm andMetaGeneAnnotator programs (Besemer, J. and Borodovsky, M. 1999. NucleicAcids Res., 27: 3911-3920; Noguchi, H. et al., 2008. DNA Res., 15:387-396). Protein homology searches were carried out with BLASTP program(Alschul, S.F. et al., 1997. Nucleic Acids Res., 25: 3389-33402) usingthe NCBI non-redundant protein sequences database. Protein conserveddomains were predicted using NCBI specialized BLAST (Marchler-Bauer, A.et al., 2007. Nucleic Acids Res. 35: 237-240). orfs whose productspresented homology with the same protein(s) are indicated with the samenumber added of a lowercase letter, in FIG. 2. Identification ofputative transfer RNA genes (tRNA) was carried out using the tRNAscan-SEprogam (Lowe, T. M. et al., 1997. Nucleic Acids Res., 25: 955-964).

FIGS. 3A-B provide the results of spot tests that assessed the activityof the bacteriophage F168/08 against 105 Enterococcus faecalis (A) and56 Enterococcus faecium (B) strains isolated from clinical samples. Eachspot consisted of 5 μl of bacteriophage suspension with the indicatedtiters, prepared from a CsCl purified lysate. Sensitivity to the phageis represented as a scale ranging from turbid (+) to clear (++++) lysishalos. Resistance to phage infection is indicated as (−).

7.3 Example 2 Bacteriophage F170/08

Comparison of the putative ORFs of the bacteriophage F170/08 genome withthe sequences in the NCBI nucleotide database revealed that about 94% ofits genome was highly similar to that of Enterococcus phage ΦEF24C, withindividual ORF identities ranging from 80 to 100%. The F170/08 ORFs,their encoded amino acid sequences and known homologous proteins areprovided in FIG. 5. Prediction of orfs was performed by integrating theresults obtained with GeneMark.hmm and MetaGeneAnnotator programs(Besemer, J. and Borodovsky, M. 1999. Nucleic Acids Res., 27: 3911-3920;Noguchi, H. et al., 2008. DNA Res., 15: 387-396). Protein homologysearches were carried out with BLASTP program (Alschul, S. F. et al.,1997. Nucleic Acids Res., 25: 3389-33402) using the NCBI non-redundantprotein sequences database. Protein conserved domains were predictedusing NCBI specialized BLAST (Marchler-Bauer, A. et al., 2007. NucleicAcids Res. 35: 237-240). ° ifs whose products presented homology withthe same protein(s) are indicated with the same number added of alowercase letter, in FIG. 5. Identification of putative transfer RNAgenes (tRNA) was carried out using the tRNAscan-SE progam (Lowe, T. M.et al., 1997. Nucleic Acids Res., 25: 955-964).

FIGS. 6A-B provide the results of spot tests that assessed the activityof the bacteriophage F170/08 against 105 Enterococcus faecalis (A) and56 Enterococcus faecium (B) strains isolated from clinical samples. Eachspot consisted of 5 μl of bacteriophage suspension with the indicatedtiters, prepared from a CsCl purified lysate. Sensitivity to the phageis represented as a scale ranging from turbid (+) to clear (++++) lysishalos. Resistance to phage infection is indicated as (−).

7.4 Example 3 Bacteriophage F770/05

Comparison of the putative ORFs of the bacteriophage F770/05 genome withthe sequences in the NCBI nucleotide database revealed that only smallportions of the genome (≦1%) exhibited homology with known sequences.The F170/05 ORFs, their encoded amino acid sequences and knownhomologous proteins are provided in FIG. 8. Prediction of ° ifs wasperformed by integrating the results obtained with GeneMark.hmm andMetaGeneAnnotator programs (Besemer, J. and Borodovsky, M. 1999. NucleicAcids Res., 27: 3911-3920; Noguchi, H. et al., 2008. DNA Res., 15:387-396). Protein homology searches were carried out with BLASTP program(Alschul, S. F. et al., 1997. Nucleic Acids Res., 25: 3389-33402) usingthe NCBI non-redundant protein sequences database. Protein conserveddomains were predicted using NCBI specialized BLAST (Marchler-Bauer, A.et al., 2007. Nucleic Acids Res. 35: 237-240).

FIG. 9 provides the results of spot tests that assessed the activity ofthe bacteriophage F170/05 against 100 Pseudomonas aeruginosa strainsisolated from clinical samples. Each spot consisted of 5 μl ofbacteriophage suspension with the indicated titers, prepared from a CsClpurified lysate. Sensitivity to the phage is represented as a scaleranging from turbid (+) to clear (++++) lysis halos. Resistance to phageinfection is indicated as (−).

7.5 Example 4 Bacteriophage F197/08

Comparison of the putative ORFs of the bacteriophage F197/08 genome withthe sequences in the NCBI nucleotide database revealed that the genomewas highly homologous with multiple staphylococcal phage genomes. Inparticular, about 90% of the F197/08 genome is highly similar to that ofStaphylococcus bacteriophage phiSauS-IPLA35, with individual ORFidentities ranging from 80 to 98%. The F197/08 ORFs, their encoded aminoacid sequences and known homologous proteins are provided in FIG. 11.Prediction of orfs was performed by integrating the results obtainedwith GeneMark.hmm and MetaGeneAnnotator programs (Besemer, J. andBorodovsky, M. 1999. Nucleic Acids Res., 27: 3911-3920; Noguchi, H. etal., 2008. DNA Res., 15: 387-396). Protein homology searches werecarried out with BLASTP program (Alschul, S.F. et al., 1997. NucleicAcids Res., 25: 3389-33402) using the NCBI non-redundant proteinsequences database. Protein conserved domains were predicted using NCBIspecialized BLAST (Marchler-Bauer, A. et al., 2007. Nucleic Acids Res.35: 237-240). offs whose products presented homology with the sameprotein(s) are indicated with the same number added of a lowercaseletter, in FIG. 11.

FIG. 12 provides the results of spot tests that assessed the activity ofthe bacteriophage F197/08 against 100 Staphylococcus aureus strainsisolated from clinical samples. Each spot consisted of 5 μl ofbacteriophage suspension with the indicated titers, prepared from a CsClpurified lysate. Sensitivity to the phage is represented as a scaleranging from turbid (+) to clear (++++) lysis halos. Resistance to phageinfection is indicated as (−).

7.6 Example 5 Bacteriophage F86/06

Comparison of the putative ORFs of the bacteriophage F86/06 genome withthe sequences in the NCBI nucleotide database revealed that the genomewas highly homologous with multiple staphylococcal phage genomes. Inparticular, about 80% of the F86/06 genome is highly similar to that ofStaphylococcus bacteriophage tp310-1, with individual ORF identitiesranging from 85 to 100%. The F86/06 ORFs, their encoded amino acidsequences and known homologous proteins are provided in FIG. 14.Prediction of orfs was performed by integrating the results obtainedwith GeneMark.hmm and MetaGeneAnnotator programs (Besemer, J. andBorodovsky, M. 1999. Nucleic Acids Res., 27: 3911-3920; Noguchi, H. etal., 2008. DNA Res., 15: 387-396). Protein homology searches werecarried out with BLASTP program (Alschul, S. F. et al., 1997. NucleicAcids Res., 25: 3389-33402) using the NCBI non-redundant proteinsequences database. Protein conserved domains were predicted using NCBIspecialized BLAST (Marchler-Bauer, A. et al., 2007. Nucleic Acids Res.35: 237-240).

FIG. 15 provides the results of spot tests that assessed the activity ofthe bacteriophage F86/06 against 100 Staphylococcus aureus strainsisolated from clinical samples. Each spot consisted of 5 μl ofbacteriophage suspension with the indicated titers, prepared from a CsClpurified lysate. Sensitivity to the phage is represented as a scaleranging from turbid (+) to clear (++++) lysis halos. Resistance to phageinfection is indicated as (−).

7.7 Example 6 Bacteriophage F87s/06

Comparison of the putative ORFs of the bacteriophage F87s/06 genome withthe sequences in the NCBI nucleotide database revealed that the genomewas highly homologous with multiple staphylococcal phage genomes. Inparticular, about 82% of the F87s/06 genome is highly similar to that ofStaphylococcus bacteriophage ΦNM3, with individual ORF identitiesranging from 90 to 100%. The F86/06 ORFs, their encoded amino acidsequences and known homologous proteins are provided in FIG. 17.Prediction of ° ifs was performed by integrating the results obtainedwith GeneMark.hmm and MetaGeneAnnotator programs (Besemer, J. andBorodovsky, M. 1999. Nucleic Acids Res., 27: 3911-3920; Noguchi, H. etal., 2008. DNA Res., 15: 387-396). Protein homology searches werecarried out with BLASTP program (Alschul, S. F. et al., 1997. NucleicAcids Res., 25: 3389-33402) using the NCBI non-redundant proteinsequences database. Protein conserved domains were predicted using NCBIspecialized BLAST (Marchler-Bauer, A. et al., 2007. Nucleic Acids Res.35: 237-240). coifs whose products presented homology with the sameprotein(s) are indicated with the same number added of a lowercaseletter, in FIG. 17.

FIG. 18 provides the results of spot tests that assessed the activity ofthe bacteriophage F87s/06 against 100 Staphylococcus aureus strainsisolated from clinical samples. Each spot consisted of 5 μl ofbacteriophage suspension with the indicated titers, prepared from a CsClpurified lysate. Sensitivity to the phage is represented as a scaleranging from turbid (+) to clear (++++) lysis halos. Resistance to phageinfection is indicated as (−).

7.8 Example 7 Bacteriophage F91a/06

Comparison of the putative ORFs of the bacteriophage F91a/06 genome withthe sequences in the NCBI nucleotide database revealed that the genomewas highly homologous with multiple staphylococcal phage genomes. Inparticular, about 82% of the F91a/06 genome is highly similar to that ofStaphylococcus bacteriophage ONM3, with individual ORF identitiesranging from 86 to 99%. The F91a/06 ORFs, their encoded amino acidsequences and known homologous proteins are provided in FIG. 20.Prediction of orfs was performed by integrating the results obtainedwith GeneMark.hmm and MetaGeneAnnotator programs (Besemer, J. andBorodovsky, M. 1999. Nucleic Acids Res., 27: 3911-3920; Noguchi, H. etal., 2008. DNA Res., 15: 387-396). Protein homology searches werecarried out with BLASTP program (Alschul, S. F. et al., 1997. NucleicAcids Res., 25: 3389-33402) using the NCBI non-redundant proteinsequences database. Protein conserved domains were predicted using NCBIspecialized BLAST (Marchler-Bauer, A. et al., 2007. Nucleic Acids Res.35: 237-240). orfs whose products presented homology with the sameprotein(s) are indicated with the same number added of a lowercaseletter, in FIG. 20.

FIG. 21 provides the results of spot tests that assessed the activity ofthe bacteriophage F91a/06 against 100 Staphylococcus aureus strainsisolated from clinical samples. Each spot consisted of 5 μl ofbacteriophage suspension with the indicated titers, prepared from a CsClpurified lysate. Sensitivity to the phage is represented as a scaleranging from turbid (+) to clear (++++) lysis halos. Resistance to phageinfection is indicated as (−).

7.9 Example 8 Bacteriophage F1245/05

F1245/05 ORFs, their encoded amino acid sequences and known homologousproteins are provided in FIGS. 23A-I. Prediction of orfs was performedby integrating the results obtained with GeneMark.hmm andMetaGeneAnnotator programs (Besemer, J. and Borodovsky, M. 1999. NucleicAcids Res., 27: 3911-3920; Noguchi, H. et al., 2008. DNA Res., 15:387-396). Protein homology searches were carried out with BLASTP program(Alschul, S. F. et al., 1997. Nucleic Acids Res., 25: 3389-33402) usingthe NCBI non-redundant protein sequences database. Protein conserveddomains were predicted using NCBI specialized BLAST (Marchler-Bauer, A.et al., 2007. Nucleic Acids Res. 35: 237-240). Protein function waspredicted based on genome localization of the corresponding gene and onthe presence of putative transmembrane domains of the encoded product(TMHMM server v. 2.0; Krogh, A. et al., 2001. J. Mol. Biol., 305:567-580.

FIGS. 24 A-E provides the results of spot tests that assessed theactivity of the bacteriophage F1245/05 against 100 Acinetobacterbaumanni strains isolated from clinical samples. Each spot consisted of5 μl of bacteriophage suspension with the indicated titers, preparedfrom a CsCl purified lysate. Sensitivity to the phage is represented asa scale ranging from turbid (+) to clear (++++) lysis halos. Resistanceto phage infection is indicated as (−).

What is claimed is:
 1. A method of treating or reducing the incidence ofa Pseudomonas aeruginosa bacterial infection in a subject in needthereof, said method comprising administering to said subject aneffective amount of a pharmaceutical composition comprising a purifiedbacteriophage having a nucleic acid sequence of SEQ ID NO:3 and apharmaceutically acceptable carrier.
 2. The method of claim 1, whereinthe infection is a nosocomial infection.
 3. The method of claim 1further comprising administering to said subject an antibiotic fortreating infection by Pseudomonas aeruginosa.
 4. The method of claim 1,wherein said pharmaceutical composition further comprises one or moreadditional bacteriophage known to have antibacterial or antimicrobialactivity against Pseudomonas aeruginosa, and/or one or more additionalbacteriophage known to have antibacterial or antimicrobial activityagainst bacteria other than Pseudomonas aeruginosa.
 5. The method ofclaim 1, wherein the pharmaceutical composition is administeredtopically.
 6. The method of claim 1, wherein the infection is aninfection of the skin.
 7. The method of claim 6, wherein thepharmaceutical composition is administered topically.
 8. The method ofclaim 1, wherein the infection is an infection associated with a burninjury or diabetic foot ulcer.
 9. The method of claim 8, wherein thepharmaceutical composition is administered topically.
 10. The method ofclaim 1, wherein the infection is an infection of the lungs.
 11. Themethod of claim 10, wherein the pharmaceutical composition isadministered by inhalation.
 12. The method of claim 11, whereinadministration by inhalation uses a pump, a spray or a nebulizer. 13.The method of claim 11, wherein the pharmaceutical composition foradministration by inhalation comprises a dry powder inhaler or anaerosol spray.
 14. The method of claim 1, wherein the infection is aninfection of the urinary tract or kidneys.
 15. The method of claim 14,wherein the pharmaceutical composition is administered by a catheter.16. The method of claim 1, wherein the subject is a mammal.